NOVEL a4B7 THIOETHER PEPTIDE DIMER ANTAGONISTS

ABSTRACT

The invention relates to thioether monomer and dimer peptide molecules which inhibit binding of α4β7 to the mucosal addressing cell adhesion molecule (MAdCAM) in vivo.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/614,047, filed Jun. 5, 2017; which is a Continuation of U.S.application Ser. No. 14/714,198, filed May 15, 2015, now U.S. Pat. No.9,714,270, issued Jul. 25, 2017; which claims priority to U.S.Provisional Application No. 61/994,699, filed on May 16, 2014, U.S.Provisional Application No. 61/994,717, filed on May 16, 2014, U.S.Provisional Application No. 62/058,499, filed on Oct. 1, 2014, and U.S.Provisional Application No. 62/058,501, filed on Oct. 1, 2014, all ofwhich are incorporated by reference herein in their entireties.

SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is PRTH_010_04US_ST25.txt. The text file is 276 KB,was created on Jul. 19, 2018, and is being submitted electronically viaEFS-Web.

FIELD OF THE INVENTION

The present invention relates to the field of engineered peptides, andto the field of peptides that bind to integrins. In particular, thepresent invention relates to thioether peptides (e.g. thioether peptidemonomers and dimers) that inhibit binding of α4β7 to the mucosaladdressin cell adhesion molecule (MAdCAM) in vitro, and show highselectivity against α4β1 binding.

BACKGROUND OF THE INVENTION

Integrins are noncovalently associated α/β heterodimeric cell surfacereceptors involved in numerous cellular processes ranging from celladhesion and migration to gene regulation (Dubree, et al., Selectiveα4β7 Integrin Antagonist and Their Potential as Anti-inflammatoryAgents, J. Med. Chem. 2002, 45, 3451-3457). Differential expression ofintegrins can regulate a cell's adhesive properties, allowing differentleukocyte populations to be recruited to specific organs in response todifferent inflammatory signals. If left unchecked, the integrin-mediatedadhesion process can lead to chronic inflammation and autoimmunedisease.

The α4 integrins, α4β1 and α4β7, play essential roles in lymphocytemigration throughout the gastrointestinal tract. They are expressed onmost leukocytes, including B and T lymphocytes, where they mediate celladhesion via binding to their respective primary ligands, vascular celladhesion molecule (VCAM), and mucosal addressin cell adhesion molecule(MAdCAM), respectively. The proteins differ in binding specificity inthat VCAM binds both α4β1 and to a lesser extent α4β7, while MAdCAM ishighly specific for α4β7. In addition to pairing with the α4 subunit,the β7 subunit also forms a heterodimeric complex with αE subunit toform αEβ7, which is primarily expressed on intraepithelial lymphocytes(IEL) in the intestine, lung and genitourinary tract. αEβ7 is alsoexpressed on dendritic cells in the gut. The αEβ7 heterodimer binds toE-cadherin on the epithelial cells. The IEL cells are thought to providea mechanism for immune surveillance within the epithelial compartment.Therefore, blocking αEβ7 and α4β7 together may be a useful method fortreating inflammatory conditions of the intestine.

Inhibitors of specific integrins-ligand interactions have been showneffective as anti-inflammatory agents for the treatment of variousautoimmune diseases. For example, monoclonal antibodies displaying highbinding affinity for α4β7 have displayed therapeutic benefits forgastrointestinal auto-inflammatory/autoimmune diseases, such as Crohn'sdisease, and ulcerative colitis (Id). However, these therapiesinterfered with α4β1 integrin-ligand interactions thereby resulting indangerous side effects to the patient. Therapies utilizing smallmolecule antagonists have shown similar side effects in animal models,thereby preventing further development of these techniques.

Accordingly, there is a need in the art for integrin antagonistmolecules having high affinity for the α4β7 integrin and highselectivity against the α4β1 integrin, as a therapy for variousgastrointestinal autoimmune diseases.

Such integrin antagonist molecules are disclosed herein.

SUMMARY OF THE INVENTION

The present invention has been developed in response to the presentstate of the art, and in particular, in response to the problems andneeds in the art that have not yet been fully solved by currentlyavailable integrin antagonists that are selective for α4β7. Thus, incertain aspects, the present invention provides α4β7 antagonistthioether peptide monomers and dimers for use as anti-inflammatoryand/or immunosuppressive agents. Further, the present invention providesα4β7 antagonist thioether peptides (e.g. monomers and dimers for use intreating a condition that is associated with a biological function ofα4β7 or on cells or tissues expressing MAdCAM.

Aspects of the invention relate to a novel class of cyclized, thioetherpeptidic compounds exhibiting integrin antagonist activity, namely,exhibiting high specificity for α4β7 integrin. In certain embodiments,each peptide of the present invention comprises a downstream natural orunnatural amino acid and an upstream modified amino acid or aromaticgroup that are capable of bridging to form a cyclized structure througha thioether bond. Peptides of the present invention demonstrateincreased stability when administered orally as a therapeutic agent. Thepeptides of the present invention further provide increased specificityand potency as compared to analogs that are cyclized through a bondother than a thioether bond, e.g., a disulfide bond.

In certain embodiments, cyclized, thioether peptidic compoundsexhibiting integrin antagonist activity are monomer peptides. Inparticular embodiments, the compounds of the present invention comprisedimerized peptides, each subunit of the dimer forming a cyclizedstructure through a thioether bond. The thioether cyclization featureprovides the peptides of the present invention increased stability,specificity, and potency as compared to analogs that are cyclizedthrough a bond other than a thioether bond, e.g., a disulfide bond. Insome embodiments, dimerization of thioether peptide monomers furtherprovides for increased specificity and potency as compared monomeranalogs.

In one embodiment, the invention provides a peptide molecule comprisinga structure of Formula (V):

(Formula (V) (SEQ ID NO: 49)Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Xaa¹²-Xaa¹³-Xaa¹⁴

or a pharmaceutically acceptable salt thereof, wherein the peptidecomprises a thioether bond between Xaa⁴ and Xaa¹⁰, and wherein:

Xaa¹ is absent, or Xaa¹ is any amino acid;

Xaa² is absent, or Xaa² is any amino acid;

Xaa³ is absent, or Xaa³ is any amino acid;

Xaa⁴ is an amino acid, aliphatic acid, alicyclic acid, or modified2-methyl aromatic acid having a side chain with one or two carbons, andcapable of forming a thioether bond with Xaa¹⁰;

Xaa⁵ is selected from the group consisting of N(alpha)-Me-Arg, Arg,HomoArg, Dap, Dab, Arg-Me-sym, Arg-Me-asym, 4-Guan, Cit, Cav, N-Me-Lys,Phe(4-quanidino), Phe(4-carbamoyl amino), Phe(4-NH₂), N-Me-HomoArg, Tyr,His, and suitable isostere replacements;

Xaa⁶ is selected from the group consisting of Ser, Gly, Thr, Ile, andsuitable isostere replacements;

Xaa⁷ is selected from the group consisting of Asp, N-Me-Asp, Asp(OMe),D-Asp, and suitable isostere replacements;

Xaa⁸ is selected from the group consisting of Thr, Gln, Ser, Asp, Pro,Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu, Val, Tyr, Trp, Leu, Met,HomoLeu, Nle, and N-Methyl amino acids including N-Me-Thr;

Xaa⁹ is selected from the group consisting of Gln, Asn, Asp, Pro, Gly,Ala, Phe, Leu, Glu, Ile, Val, HLeu, n-Butyl Ala, n-Pentyl Ala, n-HexylAla, Nle, cyclobutyl-Ala, Cpa, Aoc, N-Me-Leu, and suitable isosterereplacements;

Xaa¹⁰ is selected from the group consisting of Cys, N-Me-Cys, D-Cys,HCys, Pen, D-Pen, and Pen(=O);

Xaa¹¹ is absent or is selected from the group consisting of: Trp, Phe,2-Nal, 1-Nal, Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3), Phe (4-tBu),Bip, Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic,b-homo-Trp, D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl),Phe(4-carbomyl), Phe(3-Carbomyl), Phe (2-carbomyl), Tyr(Me), HomoPhe,N-Me-Phe, N-Me-Tyr, Ser, Sar, Dihydro Trp, Ile, Leu, Ser, Arg, Thr, Sar,and Ser, aromatic amino acids, substituted aromatic amino acids, Gly,Gln, Asn, Asp, Ala, Ile, Leu, Val, Met, Thr, Lys, Trp, Tyr, His, Glu,Ser, Arg, Pro, Phe, Sar, 1-Nal, 2-Nal, D-1-Nal, D-2-Nal, HPhe, D-Phe,D-Tyr, Phe(4-F), O-Me-Tyr, dihydro-Trp, Dap, Dab, Dab(Ac), Orn, D-Orn,N-Me-Orn, N-Me-Dap, D-Dap, D-Dab, Bip, Ala(3,3diphenyl), Biphenyl-Ala,aromatic ring substituted Phe, aromatic ring substituted Trp, aromaticring substituted His, hetero aromatic amino acids, N-Me-Lys,N-Me-Lys(Ac), 4-Me-Phe, Phe(4tBu), Phe(4-OMe), Phe(4-COOH),Phe(2-carbomyl), Phe(3-carbomyl), Phe(CF3), Phe(2,4-diCl),Phe(3,4-diCl), Aic, N-Me-Tyr, N-Me-Phe, Tic, Phe(4CF3), Bpa, Phe(3-Me),Phe(2-Me), Phe(2-CF3), β-Me-Phe, and corresponding D-amino acids andsuitable isostere replacements;

Xaa¹² is absent or selected from the group consisting of aromatic aminoacids, substituted aromatic amino acids, Glu, D-Glu, HomoGlu,Beta-Homo-Glu, Asp, D-HomoGlu, Amide, Lys, COOH, CONH₂, Gln, Pro, Gly,His, Ala, Ile, Phe, Arg, Leu, Val, Tyr, Trp, Met, Gla, Ser, Asn, D-Glu,β-HGlu, 2-Nal, 1-Nal, D-Asp, Bip, β-HPhe, β-Glu, D-Tyr, D-Phe, D-Lys,Dap, Dab, Orn, D-Orn, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me Lys, D-Dap,D-Dab, D-His, F(4-COOH), Tic, D-Trp, D-Leu, D-Arg, D-Thr, N-Me-Glu,N-Me-Asp, alpha-H-Glu, suitable isosteres, and corresponding D-aminoacids;

Xaa¹³ is absent or any amino acid; and

Xaa¹⁴ is absent or any amino acid;

wherein if the peptide molecule is a peptide dimer or subunit thereof,then Xaa¹⁴ is absent or selected from the group consisting of: any aminoacid with an amine side chain, Lys, D-Lys, N-Me-Lys, D-N-Me-Lys, Orn,N-Me-Orn, Dab, N-Me-Dab, Dap, N-Me-Dap, Homo-Lys, D-Dap, D-Dab, D-Orn,Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met,Glu, Ser, Asn, Gla, Cys, HomoCys, COOH, CONH₂, suitable isosteres,corresponding D-amino acids, and corresponding N-Methyl amino acids, andwherein the peptide molecule comprises a thioether bond between Xaa⁴ andXaa¹⁰.

In particular embodiments, Xaa¹, Xaa² and Xaa³ are absent. In certainembodiments, Xaa⁴ is a 2-methylbenzoyl moiety. In certain embodiments,Xaa⁵ is 2-Me-Arg. In particular embodiments, Xaa⁸ is selected from thegroup consisting of Thr, Gln, Ser, Asp, Gly, His, Ala, Ile, Phe, Lys,Arg, Asn, Glu, Val, Tyr, Trp, Leu, Met, HomoLeu, Nle, and N-Methyl aminoacids including N-Me-Thr. In particular embodiments, Xaa⁹ is selectedfrom the group consisting of Gln, Asn, Asp, Gly, Ala, Phe, Leu, Glu,Ile, Val, HLeu, n-Butyl Ala, n-Pentyl Ala, n-Hexyl Ala, Nle,cyclobutyl-Ala, Cpa, Aoc, N-Me-Leu, and suitable isostere replacements.In certain embodiments, Xaa¹⁴ is selected from the group consisting of:Lys, D-Lys, N-Me-Lys, D-N-Me-Lys, Orn, Dab, Dap, Homo-Lys, D-Dap, D-Dab,Cys, HomoCys, Pen, or D-Orn. In particular embodiments, Xaa¹⁴ isselected from the group consisting of: D-Lys, N-Me-Lys, and D-N-Me-Lys.In certain embodiments, the peptide molecule comprisesN(alpha)methylation of at least one position selected from the groupconsisting of Xaa³, Xaa⁵, Xaa⁷-Xaa⁹, and Xaa¹¹-Xaa¹³. In certainembodiments, the peptide molecule comprises acylation for at least oneposition selected from the group consisting of Xaa¹-Xaa³ andXaa¹¹-Xaa¹⁴.

In a related embodiment, the invention includes a peptide moleculecomprising a structure of Formula (VI) (SEQ ID NO: 387):

(Formula VI) Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹- Xaa¹⁰-Xaa¹¹

or a pharmaceutically acceptable salt thereof, wherein

Xaa¹ is a 2-Me-benzoyl group capable of forming a thioether bond withXaa⁷;

Xaa² is selected from the group consisting of N(alpha)-Me-Arg, Arg,HArg, Dap, Dab, Arg-Me-sym, Arg-Me-asym, 4-Guan, Cit, Cav, and suitableisostere replacements;

Xaa³ is selected from the group consisting of Ser, Gly, and suitableisostere replacements;

Xaa⁴ is selected from the group consisting of Asp, N-Me-Asp, Asp(OMe),D-Asp, and a suitable isostere replacements;

Xaa⁵ is selected from the group consisting of Thr, Gln, Ser, Asp, Pro,Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu, Val, Tyr, Trp, Leu, Met,and N-Methyl amino acids including N-Me-Thr, and suitable isosterereplacements;

Xaa⁶ is selected from the group consisting of Gln, Asn, Asp, Pro, Gly,Ala, Phe, Leu, Glu, Ile, Val, HLeu, n-Butyl Ala, n-Pentyl Ala, n-HexylAla, Nle, cyclobutyl-Ala, N-Me-Leu, and suitable isostere replacements;

Xaa⁷ is selected from the group consisting of Cys, N-Me-Cys, D-Cys,HCys, Pen, and D-Pen;

Xaa⁸ is selected from the group consisting of absent, Gly, Gln, Asn,Asp, Ala, Ile, Leu, Val, Met, Thr, Lys, Trp, Tyr, His, Glu, Ser, Arg,Pro, Phe, Sar, 1-Nal, 2-Nal, HPhe, Phe(4-F), O-Me-Tyr, dihydro-Trp, Dap,Dab, Dab(Ac), Orn, D-Orn, N-Me-Orn, N-Me-Dap, D-Dap, D-Dab, Bip,Ala(3,3diphenyl), Biphenyl-Ala, aromatic ring substituted Phe, aromaticring substituted Trp, aromatic ring substituted His, hetero aromaticamino acids, N-Me-Lys, N-Me-Lys(Ac), Bpa, Phe(3-Me), Phe(2-Me),Phe(2-CF3), β-Me-Phe, 4-Me-Phe, and corresponding D-amino acids andsuitable isostere replacements;

Xaa⁹ is selected from the group consisting of absent, Glu, Amide, Lys,COOH, CONH₂, Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr,Trp, Met, Gla, Ser, Asn, D-Glu, β-HGlu, 2-Nal, 1-Nal, D-Asp, Bip,β-HPhe, β-Glu, D-Tyr, D-Lys, Dap, Dab, Orn, D-Orn, N-Me-Orn, N-Me-Dap,N-Me-Dab, N-Me Lys, D-Dap, D-Dab, Glu, N-Me-Asp, alpha-H-Glu, suitableisosteres, and corresponding D-amino acids;

Xaa¹⁰ is selected from the group consisting of absent, Gln, Pro, Gly,His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser, Asn,Gla, Dap, Dab, Orn, D-Orn, D-Lys, N-Me-Orn, N-Me-Dap, N-Me-Dab,N-Me-Lys, D-Dap, D-Dab, COOH, CONH₂, suitable isosteres, andcorresponding D-amino acids; and

Xaa¹¹ is selected from the group consisting of absent, Gln, Pro, Gly,His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser, Asn,Gla, Dap, Dab, Orn, D-Orn, D-Lys, N-Me-Orn, N-Me-Dap, N-Me-Dab,N-Me-Lys, D-Dap, D-Dab, COOH, CONH₂, suitable isosteres, andcorresponding D-amino acids, wherein the peptide further comprises athioether bond between Xaa¹ and Xaa⁷,

wherein the peptide further comprises a thioether bond between Xaa¹ andXaa⁷.

In particular embodiments, Xaa⁵ is selected from the group consisting ofThr, Gln, Ser, Asp, Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu, Val,Tyr, Trp, Leu, Met, and N-Methyl amino acids including N-Me-Thr, andsuitable isostere replacements. In particular embodiments, Xaa⁶ isselected from the group consisting of Gln, Asn, Asp, Gly, Ala, Phe, Leu,Glu, Ile, Val, HLeu, n-Butyl Ala, n-Pentyl Ala, n-Hexyl Ala, Nle,cyclobutyl-Ala, N-Me-Leu, and suitable isostere replacements. Inparticular embodiments, any of the peptide molecules of the presentinvention, further comprise a terminal modifying group selected from thegroup consisting of DIG, PEG4, PEG13, PEG25, PEG1K, PEG2K, PEG4K, PEG5K,Polyethylene glycol having molecular weight from 400 Da to 40,000 Da,IDA, Ac-IDA, ADA, Glutaric acid, Isophthalic acid, 1,3-phenylenediaceticacid, 1,4-phenylenediacetic acid, 1,2-phenylenediacetic acid, AADA,suitable aliphatic acids, suitable aromatic acids, and heteroaromaticacids. In certain embodiments, the C-terminus of the peptide moleculefurther comprises a modifying group.

In certain embodiments, the peptide molecules are monomers.

In certain embodiments, the peptide molecules are dimers. In certainembodiments, a dimer comprises two peptide molecules of the presentinvention dimerized by a linker. In particular embodiments, the linkeris selected from the group consisting of: DIG, PEG4, PEG4-biotin, PEG13,PEG25, PEG1K, PEG2K, PEG3.4K, PEG4K, PEG5K, IDA, ADA, Boc-IDA, Glutaricacid, Isophthalic acid, 1,3-phenylenediacetic acid,1,4-phenylenediacetic acid, 1,2-phenylenediacetic acid, Triazine,Boc-Triazine, IDA-biotin, PEG4-Biotin, AADA, suitable aliphatics,aromatics, heteroaromatics, and polyethylene glycol based linkers havinga molecular weight from approximately 400 Da to approximately 40,000 Da.In certain embodiments, the two peptide molecules are dimerized viatheir C-termini.

In another embodiment, the present invention includes a pharmaceuticalcomposition comprising a peptide molecule of the invention and apharmaceutically acceptable carrier, diluent or excipient. In particularembodiments, the pharmaceutical composition is formulated for oraldelivery. In certain embodiments, it further comprises an entericcoating. In certain embodiments, the enteric coating releases thepharmaceutical composition within a subject's lower gastrointestinalsystem.

In a further related embodiment, the present invention provides a methodfor treating or preventing a disease or condition that is associatedwith a biological function of integrin α4β7, the method comprisingproviding to a subject in need thereof an effective amount of a peptidemolecule of the invention or a pharmaceutical composition of theinvention. In certain embodiments, the disease or condition is aninflammatory bowel disease. In particular embodiments, the inflammatorybowel disease is ulcerative colitis or Crohn's disease. In particularembodiments, the peptide molecule inhibits binding of α4β7 to MAdCAM. Incertain embodiments, the peptide molecule or the pharmaceuticalcomposition is provided to the subject in need thereof at an intervalsufficient to ameliorate the condition. In certain embodiments, theinterval is selected from the group consisting of around the clock,hourly, every four hours, once daily, twice daily, three times daily,four times daily, every other day, weekly, bi-weekly, and monthly. Inparticular embodiments, the peptide molecule or pharmaceuticalcomposition is provided as an initial does followed by one or moresubsequent doses, and the minimum interval between any two doses is aperiod of less than 1 day, and wherein each of the doses comprises aneffective amount of the peptide molecule. In particular embodiments, theeffective amount of the peptide molecule or the pharmaceuticalcomposition is sufficient to achieve at least one of the following: a)about 50% or greater saturation of MAdCAM binding sites on α4β7 integrinmolecules; b) about 50% or greater inhibition of α4β7 integrinexpression on the cell surface; and c) about 50% or greater saturationof MAdCAM binding sites on α4β7 molecules and about 50% or greaterinhibition of α4β7 integrin expression on the cell surface, wherein i)the saturation is maintained for a period consistent with a dosingfrequency of no more than twice daily; ii) the inhibition is maintainedfor a period consistent with a dosing frequency of no more than twicedaily; or iii) the saturation and the inhibition are each maintained fora period consistent with a dosing frequency of no more than twice daily.In certain embodiments, the peptide molecule is administered orally,parenterally, or topically.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the manner in which the above-recited and other featuresand advantages of the invention are obtained will be readily understood,a more particular description of the invention briefly described abovewill be rendered by reference to specific embodiments thereof which areillustrated in the appended drawings. Understanding that these drawingsdepict only typical embodiments of the invention and are not thereforeto be considered to be limiting of its scope, the invention will bedescribed and explained with additional specificity and detail throughthe use of the accompanying drawings in which:

FIG. 1 is a schematic showing C- and N-terminal dimerization via linkermolecules according to certain representative embodiments of peptidedimers of the present invention. For example, in C-terminaldimerization, the NH₂ group may be a side chain of the C-terminal aminoacid, and in N-terminal dimerization, the NH₂ group may be an N-terminalfree amine group.

FIG. 2 is a schematic showing an integrin antagonist peptide dimer,comprising two thioether monomer subunits according to SEQ ID NO: 22,wherein the subunits are aligned and linked at their respectiveC-termini by a DIG linker moiety in accordance with a representativeembodiment of the present invention. Lowercase k indicates D-Lysine.

FIG. 3 is a schematic showing a cyclized, thioether peptide monomer ormonomer subunit of a dimer molecule according to SEQ ID NO: 1 (Formula(I)), wherein a thioether bond is formed between Xaa⁴ and Xaa¹⁰ inaccordance with a representative embodiment of the present invention.

FIG. 4 is a schematic showing a cyclized, thioether peptide monomer ormonomer subunit of a dimer molecule according to SEQ ID NO: 2 (Formula(II)), wherein Xaa¹ is a 2-methylbenzoyl moiety forming a thioether bondwith Xaa⁷ in accordance with a representative embodiment of the presentinvention. Non-limiting examples of suitable chemical moieties forsubstitution at R1-R4 are provided and discussed below.

FIG. 5 is a diagram of an illustrative linker system that may be used todimerize monomer subunits of dimer molecules of the present invention,e.g., dimerization through a sulfhydryl group. FIG. 5 shows a pair ofintegrin antagonist monomer subunits wherein the subunits are alignedand linked at their respective C-termini by a linker that connects twosulfur-containing amino-acids to form a peptide dimer linkingsulfhydryl-to-sulfhydryl crosslinking of the present invention, whereinX₁ and X₂ are H or Me; and the linker (Y) is defined as shown. Inparticular embodiments, the linker (Y) can comprise homobifunctionalmaleimide crosslinkers, di-halide, 1,2-Bis(bromomomethyl)benzene,1,2-Bis(chloromomethyl)benzene, 1,3-Bis(bromomomethyl)benzene,1,3-Bis(chloromomethyl)benzene, 1,4-Bis(bromomomethyl)benzene,1,4-Bis(chloromomethyl)benzene 3,3′-Bis-bromomethyl-biphenyl, or2,2′-Bis-bromomethyl-biphenyl. Certain haloacetyl crosslinkers containan iodoacetyl or a bromoacetyl groups. In certain embodiments, thesehomobifunctional linkers may contain spacers, e.g., comprising a PEG oran aliphatic chain.

FIG. 6 is a chart demonstrating potency and stability data in simulatedintestinal fluids (SIF) for various thioether peptide dimer compoundsaccording to SEQ ID NO: 23 and Formula (II) in accordance with variousnon-limiting representative embodiment of the present invention. Lowercase letters indicate D-amino acids.

FIG. 7 is a chart demonstrating potency data of various peptide monomercompounds according to Formula II in accordance with variousnon-limiting representative embodiments of the present invention.

FIG. 8 is a chart demonstrating stability data in simulated intestinalfluids (SIF) for various peptide monomer compounds according to Formula(II) in accordance with various non-limiting representative embodimentof the present invention.

SEQUENCE IDENTIFIERS

The amino acid sequences listed in the accompanying sequence listing areshown using three letter code for amino acids, as defined in 37 C.F.R.1.822. Sequences of monomer peptide molecules or the monomer subunits ofdimer molecules are shown.

In the accompanying sequence listing:

SEQ ID NO: 1 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (I).

SEQ ID NO: 2 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (II).

SEQ ID NOs: 1-32 show amino acid sequences of illustrative thioethermonomer peptides or thioether peptide subunits that are dimerized toform various thioether dimer compounds in accordance with the presentinvention, wherein these sequences have been substituted with anN(alpha)-Me-Arg.

SEQ ID NO: 33 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (I-1).

SEQ ID NO: 34 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (I-2).

SEQ ID NO: 35 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (I-3).

SEQ ID NO: 36 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (I-A).

SEQ ID NO: 37 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (I-B).

SEQ ID NO: 38 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (I-C).

SEQ ID NO: 39 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (I-D).

SEQ ID NO: 40 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (I-E).

SEQ ID NO: 41 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (I-F).

SEQ ID NO: 42 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (I-G).

SEQ ID NO: 43 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (I-H).

SEQ ID NO: 44 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (I-I).

SEQ ID NO: 45 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (II-A).

SEQ ID NO: 46 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (III).

SEQ ID NO: 47 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (IV).

SEQ ID NO: 48 shows a monomer peptide molecule or a peptide subunit of adimer molecule representing various thioether peptides or peptidesubunits of Formula (A).

SEQ ID NO:49 shows a monomeric peptide molecule or a peptide subunit ofa dimer molecule representing various thioether peptides or peptidesubunits of Formula (V)

SEQ ID NO:50 shows a monomeric peptide molecule or a peptide subunit ofa dimer molecule representing various thioether peptides or peptidesubunits of Formula (VI).

SEQ ID NOs: 1, 2, 5, 6, 9-21 and 25-32 show various amino acid sequencesof illustrative thioether peptides that may be acylated at theirN-terminus using one of the acylating organic compounds and methodsdisclosed herein, including but not limited to cyclopropylacetic acid,4-Fluorobenzoic acid, 4-fluorophenylacetic acid, 3-Phenylpropionic acid,Succinic acid, Glutaric acid, Cyclopentane carboxylic acid,3,3,3-trifluoropropeonic acid, and 3-Fluoromethylbutyric acid.

SEQ ID NOs: 1-21 and 25-32 show amino acid sequences of illustrativemonomer subunits that may be dimerized at either their N- orC-terminuses to form various thioether dimer compounds in accordancewith the present invention.

SEQ ID NOs: 22-24 show amino acid sequences of monomer subunits that maybe dimerized at their C-terminuses to form various thioether dimercompounds in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, integrins are heterodimers that function as celladhesion molecules. The α4 integrins, α4β1 and α4β7, play essentialroles in lymphocyte migration throughout the gastrointestinal tract.They are expressed on most leukocytes, including B and T lymphocytes,monocytes, and dendritic cells, where they mediate cell adhesion viabinding to their respective primary ligands, namely vascular celladhesion molecule (VCAM) and mucosal addressin cell adhesion molecule(MAdCAM). VCAM and MAdCAM differ in binding specificity, in that VCAMbinds both α4β1 and α4β7, while MAdCAM is highly specific for α4β7.

The present invention relates generally to thioether peptides (e.g.peptide monomers and dimers) that have been shown to have integrinantagonist activity. In particular, the present invention relates tovarious peptides that form cyclized structures through thioether bonds.In certain embodiments, the thioether bonds are cyclized via covalentbonds formed between an upstream amino acid or aromatic acid group, anda downstream sulfur containing amino acid or isostere thereof.Surprisingly, thioether bonds formed when the upstream amino acid oraromatic acid group is 2-methylbenzoyl show superior potency. In someembodiments, thioether peptides comprising 2-methylbenzoyl possesssuperior potency as compared to thioether peptides not comprising2-methylbenzoyl. Some aspects of the present invention contemplate thatthioether peptide integrin inhibitors comprising 2-methylbenzoyl showsuperior potency compared to non-cyclized integrin peptide inhibitors.In some embodiments, thioether peptide integrin inhibitors comprising2-methylbenzoyl show superior potency compared to other integrin peptideinhibitors that do not include this moiety. As used herein, “superiorpotency” will be understood by those of skill in the art to mean agreater, higher, better, or improved potency.

Differences in the expression profiles of VCAM and MAdCAM provide themost convincing evidence of their role in inflammatory diseases. Bothare constitutively expressed in the gut; however, VCAM expressionextends into peripheral organs, while MAdCAM expression is confined toorgans of the gastrointestinal tract. In addition, elevated MAdCAMexpression in the gut has now been correlated with severalgut-associated inflammatory diseases, including Crohn's disease,ulcerative colitis, and hepatitis C.

The thioether peptide monomer and dimer molecules of the invention maybe used in combination with other compositions and procedures for thetreatment of disease. Additionally, the monomer or dimer molecules ofthe present invention may be combined with pharmaceutically acceptableexcipients, and optionally sustained-release matrices, such asbiodegradable polymers, to form therapeutic compositions.

Definitions

As used herein, the singular forms “a,” “and” and “the” include pluralreferences unless the context clearly dictates otherwise.

When the term “comprising” is used herein, it is understood that thepresent invention also includes the same embodiments wherein the term“comprising” is substituted with “consisting essentially of” or“consisting of.”

As used in the present specification the following terms have themeanings indicated:

The term “peptide,” as used herein, refers broadly to a structurecomprising a sequence of two or more amino acids joined together bypeptide bonds. In particular embodiments, it refers to a sequence of twoor more amino acids joined together by peptide bonds. It should beunderstood that this term does not connote a specific length of apolymer of amino acids, nor is it intended to imply or distinguishwhether the polypeptide is produced using recombinant techniques,chemical or enzymatic synthesis, or is naturally occurring. The term“peptide”, as used generically herein, includes both peptide monomersand peptide dimers.

The term “monomer” as used herein may also be referred to as “peptidemonomer,” “peptide monomer molecule,” or “monomer peptide.” The term“monomer” indicates a single sequence of two or more amino acids joinedtogether by peptide bonds.

The term “dimer,” as used herein, refers broadly to a peptide comprisingtwo monomer peptide subunits (e.g., thioether monomer peptides) that arelinked at their respective C- or N-terminuses. Dimers of the presentinvention may include homodimers or heterodimers that function asintegrin antagonists. The term “dimer” may also be referred to herein toas a “peptide dimer,” “peptide dimer molecule,” “dimer peptide,” or“dimer compound.” The term “monomer peptide subunit” may also bereferred to herein as “monomer subunit,” “peptide monomer subunit,”“peptide subunit,” “peptide dimer subunit,” “dimer subunit,” “monomericsubunit,” or “subunit of a peptide dimer.”

The term “thioether,” as used herein, refers to a cyclized, covalentbond formed between an upstream amino acid or aromatic acid group, and adownstream sulfur-containing amino acid, or isostere thereof, i.e., aC—S bond.

The term “linker,” as used herein, refers broadly to a chemicalstructure that is capable of linking together two thioether monomersubunits to form a dimer.

The term “L-amino acid,” as used herein, refers to the “L” isomeric formof a peptide, and conversely the term “D-amino acid” refers to the “D”isomeric form of a peptide. The amino acid residues described herein arepreferred to be in the “L” isomeric form, however, residues in the “D”isomeric form can be substituted for any L-amino acid residue, as longas the desired functional is retained by the peptide.

Unless otherwise indicated, the term “NH₂,” as used herein, refers tothe free amino group present at the amino terminus of a polypeptide. Theterm “OH,” as used herein, refers to the free carboxy group present atthe carboxy terminus of a peptide. Further, the term “Ac,” as usedherein, refers to Acetyl protection through acylation of the N-terminusof a polypeptide. Where indicated, “NH₂” refers to a free amino groupside chain of an amino acid. Where indicated, the term “Ac,” as usedherein refers to acylation of an amino acid with NH₂ group.

The term “carboxy,” as used herein, refers to —CO₂H.

The term “isotere” or “isostere replacement,” as used herein, refers toany amino acid or other analog moiety having chemical and/or structuralproperties similar to a specified amino acid. In particular embodiments,an “isostere” or “suitable isostere” of an amino acid is another aminoacid of the same class, wherein amino acids belong to the followingclasses based on the propensity of the side chain to be in contact withpolar solvent like water: hydrophobic (low propensity to be in contactwith water), polar or charged (energetically favorable contact withwater). The charged amino acid residues include lysine (+), arginine(+), aspartate (−) and glutamate (−). Polar amino acids include serine,threonine, asparagine, glutamine, histidine and tyrosine. Thehydrophobic amino acids include alanine, valine, leucine, isoleucine,proline, phenylalanine, tryptophane, cysteine and methionine. The aminoacid glycine does not have a side chain and is hard to assign to one ofthe above classes. However, glycine is often found at the surface ofproteins, often within loops, providing high flexibility to theseregions, and an isostere may have a similar feature. Proline has theopposite effect, providing rigidity to the protein structure by imposingcertain torsion angles on the segment of the polypeptide chain.

The term “cyclized,” as used herein, refers to a reaction in which onepart of a polypeptide molecule becomes linked to another part of thepolypeptide molecule to form a closed ring, such as by forming athioether bond. In particular embodiments, peptide monomers and monomersubunits of peptide dimers of the present invention are cyclized via anintramolecular thioether bond.

The term “receptor,” as used herein, refers to chemical groups ofmolecules on the cell surface or in the cell interior that have anaffinity for a specific chemical group or molecule. Binding betweenpeptide molecules and targeted integrins can provide useful diagnostictools.

The term “integrin-related diseases,” as used herein, refer toindications that manifest as a result of integrin binding, and which maybe treated through the administration of an integrin antagonist.

The term “pharmaceutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the compounds of the present inventionwhich are water or oil-soluble or dispersible, which are suitable fortreatment of diseases without undue toxicity, irritation, and allergicresponse; which are commensurate with a reasonable benefit/risk ratio,and which are effective for their intended use. The salts can beprepared during the final isolation and purification of the compounds orseparately by reacting an amino group with a suitable acid.Representative acid addition salts include acetate, adipate, alginate,citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,camphorate, camphorsulfonate, digluconate, glycerophosphate,hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate),lactate, maleate, mesitylenesulfonate, methanesulfonate,naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate,pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate,propionate, succinate, tartrate, trichloroacetate, trifluoroacetate,phosphate, glutamate, bicarbonate, para-toluenesulfonate, andundecanoate. Also, amino groups in the compounds of the presentinvention can be quaternized with methyl, ethyl, propyl, and butylchlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamylsulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, andiodides; and benzyl and phenethyl bromides. Examples of acids which canbe employed to form therapeutically acceptable addition salts includeinorganic acids such as hydrochloric, hydrobromic, sulfuric, andphosphoric, and organic acids such as oxalic, maleic, succinic, andcitric.

The term “N(alpha)Methylation”, as used herein, describes themethylation of the alpha amine of an amino acid, also generally termedas an N-methylation.

The term “sym methylation” or “Arg-Me-sym”, as used herein, describesthe symmetrical methylation of the two nitrogens of the guanidine groupof arginine. Further, the term “asym methylation” or “Arg-Me-asym”describes the methylation of a single nitrogen of the guanidine group ofarginine.

The term “acylating organic compounds,” as used herein refers to variouscompounds with carboxylic acid functionality, which may be used toacylate the C- and/or N-termini of a peptide molecule. Non-limitingexamples of acylating organic compounds include cyclopropylacetic acid,4-Fluorobenzoic acid, 4-fluorophenylacetic acid, 3-Phenylpropionic acid,Succinic acid, Glutaric acid, Cyclopentane carboxylic acid, glutaricacid, succinic acid, 3,3,3-trifluoropropeonic acid,3-Fluoromethylbutyric acid.

All peptide sequences are written according to the generally acceptedconvention whereby the α-N-terminal amino acid residue is on the leftand the α-C-terminal is on the right. As used herein, the term“α-N-terminal” refers to the free α-amino group of an amino acid in apeptide, and the term “α-C-terminal” refers to the free α-carboxylicacid terminus of an amino acid in a peptide.

The term “amino acid” or “any amino acid” as used here refers to any andall amino acids, including naturally occurring amino acids (e.g.,α-amino acids), unnatural amino acids, modified amino acids, andnon-natural amino acids. It includes both D- and L-amino acids. Naturalamino acids include those found in nature, such as, e.g., the 23 aminoacids that combine into peptide chains to form the building-blocks of avast array of proteins. These are primarily L stereoisomers, although afew D-amino acids occur in bacterial envelopes and some antibiotics. The“non-standard,” natural amino acids are pyrrolysine (found inmethanogenic organisms and other eukaryotes), selenocysteine (present inmany noneukaryotes as well as most eukaryotes), and N-formylmethionine(encoded by the start codon AUG in bacteria, mitochondria andchloroplasts). “Unnatural” or “non-natural” amino acids arenon-proteinogenic amino acids (i.e., those not naturally encoded orfound in the genetic code) that either occur naturally or are chemicallysynthesized. Over 140 natural amino acids are known and thousands ofmore combinations are possible. Examples of “unnatural” amino acidsinclude β-amino acids (β³ and β²), homo-amino acids, proline and pyruvicacid derivatives, 3-substituted alanine derivatives, glycinederivatives, ring-substituted phenylalanine and tyrosine derivatives,linear core amino acids, diamino acids, D-amino acids, alpha-methylamino acids and N-methyl amino acids. Unnatural or non-natural aminoacids also include modified amino acids. “Modified” amino acids includeamino acids (e.g., natural amino acids) that have been chemicallymodified to include a group, groups, or chemical moiety not naturallypresent on the amino acid.

Generally, the names of naturally occurring and non-naturally occurringaminoacyl residues used herein follow the naming conventions suggestedby the IUPAC Commission on the Nomenclature of Organic Chemistry and theIUPAC-IUB Commission on Biochemical Nomenclature as set out in“Nomenclature of α-Amino Acids (Recommendations, 1974)” Biochemistry,14(2), (1975). To the extent that the names and abbreviations of aminoacids and aminoacyl residues employed in this specification and appendedclaims differ from those suggestions, they will be made clear to thereader. Some abbreviations useful in describing the invention aredefined below in the following Table 1.

TABLE 1 Abbreviations Abbreviation Definition DIG DIGlycolic acid(Linker) Dap Diaminopropionic acid Dab Diaminobutyric acid PenPenicillamine Sar Sarcosine Cit Citroline Cav Cavanine 4-Guan4-Guanidine-Phenylalanine N-Me-Arg; N(alpha)MethylationN-Methyl-Arginine Ac- Acetyl 2-Nal 2-Napthylalanine 1-Nal1-Napthylalanine Bip Biphenylalanine O—Me-Tyr Tyrosine (O—Methyl)N-Me-Lys N-Methyl-Lysine N-Me-Lys (Ac) N-Me-Acetyl (ε) Lysine Ala (3,3diphenyle) 3,3 diphenyl alanine NH₂ Free Amine CONH₂ Amide COOH Acid Phe(4-F) 4-Fluoro-Phenylanine PEG13 Bifunctional PEG linker with 13PolyEthylene Glycol units PEG25 Bifunctional PEG linker with 25PolyEthylene Glycol units PEG1K Bifunctional PEG linker withPolyEthylene Glycol Mol wt of 1000 Da PEG2K Bifunctional PEG linker withPolyEthylene Glycol Mol wt of 2000 Da PEG3.4K Bifunctional PEG linkerwith PolyEthylene Glycol Mol wt of 3400 Da PEG5K Bifunctional PEG linkerwith PolyEthylene Glycol Mol wt of 5000 Da IDA β-Ala-Iminodiacetic acid(Linker) IDA-Palm β-Ala (Palmityl)-Iminodiacetic acid HPhe HomoPhenylalanine Ahx Aminohexanoic acid DIG—OH Glycolic monoacid TriazineAmino propyl Triazine di-acid Boc-Triazine Boc-Triazine di-acidTrifluorobutyric acid Acylated with 4,4,4-Trifluorobutyric acid2-Methly-trifluorobutyric acid acylated with 2-methy-4,4,4-Butyric acidTrifluorpentanoic acid Acylated with 5,5,5-Trifluoropentnoic acid1,4-Phenylenediacetic acid para-Phenylenediacetic acid (Linker)1,3-Phenylenediacetic acid meta-Phenylenediacetic acid (Linker) DTTDithiothreotol Nle Norleucine β-HTrp β-homoTrypophane β-HPheβ-homophenylalanine Phe(4-CF₃) 4-Trifluoromethyl Phenylalanine β-Glu

β-HGlu beta-Homo-Glu

2-2-Indane 2-Aminoindane-2-carboxylic acid 1-1-Indane1-Aminoindane-1-carboxylic acid Cpa Cyclopentyl alanine Orn OrnithineAoc 2-Amono octonoic acid Cba Cyclibutyl alanine HCha homocyclohexylAlanine Cyclobutyl Cyclobutylalanine β-HPhe, B-H-K β-homophenylalanineHLeu, homo-Leu, hK, Homoleucine Gla Gama-Carboxy-Glutamic acid Tic

Phe(4CF3) Phe L-Phe(4-CF₃)—OH Phe(4-trifluoromethyl3-(4-trifluoromethyl-phenyl)propionic acid Phe(2,4-diCl)(S)-2-amino-3-(2,4-dichlorophenyl)propionic acid Phe(3,4-diCl)(S)-2-amino-3-(3,4-dichlorophenyl)propionic acid Pen(═O) Penicillaminesulfoxide Aic aminoindan-2-carboxylic acid Phe(2-carbomyl)L-2-carbamoylphenylalanine Phe(3-carbomyl) L-3-carbamoylphenylalaninePhe(4-COOH) (4-carboxy-tert-butyl)-L-phenylalanine Phe(4-Ome)(S)-4-methoxyphenylalanine Phe(4tBu)(S)-2-amino-3-(4-tert-butyl-phenyl)propionic acid Phe(4-F)4-fluoro-L-phenylalanine Glu(OMe) L-glutamic acid g-methyl esteralpha-bromobutyryl

alpha-bromopropenyl; Propionyl

alpha-bromoisobutyryl

alpha-H-E; alpha-hGlu

F(2-Me) 2-Methyl Phenylalanine 4-Benzyl

2-Benzyl

3-Benzyl

erythro-b-F-S

Threo-b-F-S

F(2-CF3) 2-Trifluoromethyl-Phenylalanine F(CF3)4-Trifluoromethyl-Phenylalinine F(4-Me); 4-Me—F 4-Methyl PhenylalanineF(3-Me) 3-Methyl Phenylalanine Alpha-hGlu HomoGlutamc acid ATC

BPA

b-Me—F

β-dimethyl-F

2-Chloro Benzoyl

N-Me-E N-Methyl Glutamic acid k(Ac) Nε-Acety-D-Lysine k(PEG8) PEG8conjugated (Nε)-D-Lys N-Me-k(Ac) N-methyl Nε-Acetyl-D-Lysine N-Me-K(Ac)N-methyl Nε-Acetyl-Lysine F(4-tBu); F(4tBu) 4-tButyl-PhenylalanineC(thioether propane) S—CH2—CH2—CH2—S l(D-L) D-leucine

Thioether Peptide Monomers and Thioether Peptide Dimers

The present invention relates generally to thioether peptides that havebeen shown to have integrin antagonist activity. In particular, thepresent invention relates to various peptides that form cyclizedstructures through thioether bonds, e.g., intramolecular thioetherbonds. Certain embodiments relate to thioether peptide monomers withintegrin antagonist activity. Some embodiments relate to thioetherpeptide dimers with integrin antagonist activity comprising hetero- orhomo-monomer thioether peptide subunits, wherein the thioether peptidesubunits are linked at either their C- or N-terminuses, e.g., as shownin FIG. 1. The cyclized structure of the peptide monomers or peptidesubunits have been shown to increase the potency, selectivity, andstability of the peptide molecules, as discussed below. A non-limiting,representative illustration of the cyclized structure of Formula (I) isshown in FIG. 3. In some embodiments, dimerizing the peptide monomerincreases potency, selectivity, and/or stability compared to anon-dimerized peptide.

In some instances, the monomer peptides further comprise C- and/orN-termini that comprise free amine (or both C- and N-termini thatcomprise free amine). Similarly, a peptide dimer may comprise one ormore C- or N-termini that comprise a free amine. Thus, a user may modifyeither terminal end to include a modifying group such as a PEGylation,e.g., a small PEGylation (e.g. PEG4-PEG13). A user may further modifyeither terminal end through acylation. For example, in some instances atleast one of the N- and C-terminus of a peptide molecule is acylatedwith an acylating organic compound selected from the group consisting of2-Me-Trifluorobutyl, Trifluoropentyl, Acetyl, Octonyl, Butyl, Pentyl,Hexyl, Palmityl, Trifluoromethyl butyric, cyclopentane carboxylic,cyclopropylacetic, 4-fluorobenzoic, 4-fluorophenyl acetic,3-Phenylpropionic acid. In some instances, peptide molecules of theinstant invention comprise both a free carboxy terminal and a free aminoterminal, whereby a user may selectively modify the peptide to achieve adesired modification. It is further understood that the C-terminalresidues of the thioether peptides, e.g., thioether monomers, disclosedherein are amides or acids, unless otherwise indicated. One having skillin the art will therefore appreciate that the thioether peptides of theinstant invention may be selectively modified, as desired.

With respect to peptide dimers, it is understood that monomer subunitsare dimerized to form thioether peptide dimer molecules in accordancewith the present teaching and as shown generally in FIGS. 1 and 2. Themonomer subunits are joined or dimerized by a suitable linker moiety, asdefined herein. Some of the monomer subunits are shown having C- andN-termini that both comprise free amine. Thus, a user may modify eitherterminal end of the monomer subunit to eliminate either the C- orN-terminal free amine, thereby permitting dimerization at the remainingfree amine. Thus, some of the monomer subunits comprise both a freecarboxy or amide at C-terminal and a free amino terminal, whereby a usermay selectively modify the subunit to achieve dimerization at a desiredterminus. One having skill in the art will therefore appreciate that themonomer subunits of the instant invention may be selectively modified toachieve a single, specific amine for a desired dimerization.

It is further understood that the C-terminal residues of the monomersubunits disclosed herein are amides, unless otherwise indicated.Further, it is understood that dimerization at the C-terminal isfacilitated by using a suitable amino acid with a side chain havingamine functionality, as is generally understood in the art. Inparticular embodiments, a linker binds to functional amine groups in theC-terminal amino acid of each of the peptide monomer subunits to form adimer. Regarding the N-terminal residues, it is generally understoodthat dimerization may be achieved through the free amine of the terminalresidue, or may be achieved by using a suitable amino acid side chainhaving a free amine, as is generally understood in the art.

In particular embodiments, dimers are dimerized through a sulfhydrylgroup, e.g., via the C-terminus of each monomer subunit of the dimer.FIG. 5 shows a pair of integrin antagonist monomer subunits wherein thesubunits are aligned and linked at their respective C-termini by alinker that connects two sulfur-containing amino-acids to form a peptidedimer linking sulfhydryl-to-sulfhydryl crosslinking of the presentinvention, wherein X₁ and X₂ are H or Me; and the linker (Y) is definedas shown. In particular embodiments, the linker (Y) can comprisehomobifunctional maleimide crosslinkers, di-halide,1,2-Bis(bromomomethyl)benzene, 1,2-Bis(chloromomethyl)benzene,1,3-Bis(bromomomethyl)benzene, 1,3-Bis(chloromomethyl)benzene,1,4-Bis(bromomomethyl)benzene, 1,4-Bis(chloromomethyl)benzene3,3′-Bis-bromomethyl-biphenyl, or 2,2′-Bis-bromomethyl-biphenyl. Certainhaloacetyl crosslinkers contain an iodoacetyl or a bromoacetyl groups.In certain embodiments, these homobifunctional linkers may containspacers, e.g., comprising a PEG or an aliphatic chain.

In some instances, N-terminal dimerization is proceeded by acylating theC-terminus using one of the acylating organic compounds and methodsdisclosed herein, including but not limited to Acetyl, cyclopropylaceticacid, 4-Fluorobenzoic acid, 4-fluorophenylacetic acid, 3-Phenylpropionicacid, Succinic acid, Glutaric acid, Cyclopentane carboxylic acid,3,3,3-trifluoropropeonic acid, and 3-Fluoromethylbutyric acid. Forexample, where a C-terminal dimerization is desired, the N-terminuses ofthe respective monomer subunits will generally acylated prior to theC-terminuses being joined by a suitable linking moiety to provide athioether dimer compound. Conversely, where an N-terminal dimerizationis desired, the C-terminuses of the respective monomer subunits may beacylated when the C-terminus comprises a free amine, the N-terminusesbeing joined by a suitable linking moiety to provide a thioether dimercompound.

The peptide monomers and dimers of the instant invention, or peptidesubunits thereof, may further comprise one or more terminal modifyinggroups. In at least one embodiment, a terminal end of a peptide ismodified to include a terminal modifying group selected from thenon-limiting group consisting of DIG, PEG4, PEG13, PEG25, PEG1K, PEG2K,PEG4K, PEG5K, Polyethylene glycol having molecular weight from 400 Da to40,000 Da, PEG having a molecular weight of 40,000 Da to 80,000 Da, IDA,ADA, Glutaric acid, Succinic acid, Isophthalic acid,1,3-phenylenediacetic acid, 1,4-phenylenediacetic acid,1,2-phenylenediacetic acid, AADA, and suitable aliphatics, aromatics,and heteroaromatics.

In certain embodiments the N- or C-terminus of the peptide monomer orpeptide dimer subunit is linked to a modifying group. In certainembodiments, the N-terminus of a peptide is modified by one to threesuitable groups, e.g., as represented by Xaa¹, Xaa², and Xaa³, e.g., ofFormula (I) or (I-A). Similarly, in certain embodiments, the C-terminusof a peptide is modified by a suitable group. For example, theC-terminus may be acylated. In some instances, the C-terminus furthercomprises a suitable linker moiety, as disclosed herein. In certainembodiments, the C-terminus comprises NH₂ or OH.

For some embodiments of peptide dimers or peptide monomers describedherein, any of Xaa¹-Xaa⁵, Xaa⁷-Xaa⁹, and Xaa¹¹-Xaa¹² areN(alpha)Methylated. Xaa⁵ may further be Arg-Me-sym or Arg-Me-asym, andXaa¹¹ may be O-Me-Tyr, N-Me-Lys(Ac), or 4-Me-Phe. The N-terminus mayfurther be acylated. In some instances, any of Xaa¹-Xaa⁴, andXaa¹¹-Xaa¹⁴ are acylated. For example, in some instances one or moreresidues at positions Xaa⁸-Xaa¹¹ are acylated with an acylating organiccompound selected from the group consisting of 2-Me-Trifluorobutyl,Trifluoropentyl, Acetyl, Octonyl, Butyl, Pentyl, Hexyl, Palmityl,Trifluoromethyl butyric, cyclopentane carboxylic, cyclopropylacetic,4-fluorobenzoic, 4-fluorophenyl acetic, and 3-Phenylpropionic acid. Insome instances one or more residues at positions Xaa¹-Xaa⁴, andXaa¹¹-Xaa¹⁴ are acylated with an acylating organic compound selectedfrom the group consisting of 2-me-Trifluorobutyl, Trifluoropentyl,Acetyl, Octonyl, Butyl, Pentyl, Hexyl, Palmityl, Lauryl, Oleoyl, andLauryl, Trifluoromethyl butyric, cyclopentane carboxylic,cyclopropylacetic, 4-fluorobenzoic, 4-fluorophenyl acetic,3-Phenylpropionic, tetrahedro-2H-pyran-4carboxylic, succinic acid, andglutaric acid. In some instances, small PEG (e.g., PEG4-PEG13) is usedas spacer before acylations.

In some embodiments of the peptide dimers, peptide dimer subunits orpeptide monomers described herein, the N-terminus further comprises asuitable linker moiety or other modifying group. In some embodiments ofpeptide monomers described herein, the N-terminus may further beacylated.

Non-limiting examples of terminal modifying groups are provided in Table2.

TABLE 2 Illustrative Terminal Modifying Groups Abbreviation DescriptionStructure DIG DIGlycolic acid,

PEG4 Bifunctional PEG linker with 4 PolyEthylene Glycol units

PEG13 PEG with 13 PolyEthylene Glycol units

PEG25 PEG with 25 PolyEthylene Glycol units

PEG1K PolyEthylene Glycol Mol wt of 1000 Da PEG2K PolyEthylene GlycolMol wt of 2000 Da PEG3.4K PolyEthylene Glycol Mol wt of 3400 Da PEG5KPolyEthylene Glycol Mol wt of 5000 Da DIG DIGlycolic acid,

IDA β-Ala-Iminodiacetic acid

Boc-IDA Boc-β-Ala-Iminodiacetic acid

Ac-IDA Acetyl-β-Ala-Iminodiacetic acid

GTA Glutaric acid

PMA Pemilic acid

AZA Azelaic acid

DDA Dodecanedioic acid

ADA Amino diacetic acid

AADA n-Acetyl amino acetic acid

PEG4-Biotin PEG4-Biotin (Product number 10199, QuantaBioDesign)

The linker moieties of the instant invention may include any structure,length, and/or size that is compatible with the teachings herein. In atleast one embodiment, a linker moiety is selected from the non-limitinggroup consisting of DIG, PEG4, PEG4-biotin, PEG13, PEG25, PEG1K, PEG2K,PEG3.4K, PEG4K, PEG5K, IDA, ADA, Boc-IDA, Glutaric acid, Isophthalicacid, 1,3-phenylenediacetic acid, 1,4-phenylenediacetic acid,1,2-phenylenediacetic acid, Triazine, Boc-Triazine, IDA-biotin,PEG4-Biotin, AADA, suitable aliphatics, aromatics, heteroaromatics, andpolyethylene glycol based linkers having a molecular weight fromapproximately 400 Da to approximately 40,000 Da or approximately 40,000Da to approximately 80,000 Da.

When the linker is IDA, ADA or any linker with free amine it can beacylated with acylating organic compound selected from the groupconsisting of 2-me-Trifluorobutyl, Trifluoropentyl, Acetyl, Octonyl,Butyl, Pentyl, Hexyl, Palmityl, Lauryl, Oleoyl, Lauryl, Trifluoromethylbutyric, cyclopentane carboxylic, cyclopropylacetic, 4-fluorobenzoic,4-fluorophenyl acetic, 3-Phenylpropionic,tetrahedro-2H-pyran-4carboxylic, succinic acid, and glutaric acid,straight chain aliphatic acids with 10 to 20 carbon units, cholic acidand other bile acids. In some instances small PEG (PEG4-PEG13), Glu, orAsp is used as spacer before acylations.

In certain embodiments, the linker connects two monomeric subunits byconnecting two sulfur containing C- or N-terminal amino acids. In someembodiments, the two sulfur containing amino acids are connected by alinker comprising a di-halide, an aliphatic chain, or a PEG. In certainembodiments, the linker connects two monomeric subunits by connectingsulfur containing C-terminal amino acids at the C-terminus of eachmonomer subunit. In some embodiments, the two sulfur containing aminoacids are connected by a linker comprising homobifunctional maleimidecrosslinkers, di-halide, 1,2-Bis(bromomomethyl)benzene,1,2-Bis(chloromomethyl)benzene, 1,3-Bis(bromomomethyl)benzene,1,3-Bis(chloromomethyl)benzene, 1,4-Bis(bromomomethyl)benzene,1,4-Bis(chloromomethyl)benzene, 3,3′-bis-bromomethyl-biphenyl, or2,2′-bis-bromomethyl-biphenyl. Particular haloacetyl crosslinkerscontain an iodoacetyl or a bromoacetyl group. These homobifunctionallinkers may contain spacers comprising PEG or an aliphatic chain.

Non-limiting examples of suitable linker moieties are provided in Table3.

TABLE 3 Illustrative Linker Moieties Abbrivation Discription StructureDIG DIGlycolic acid,

PEG4 Bifunctional PEG linker with 4 PolyEthylene Glycol units

PEG13 Bifunctional PEG linker with 13 PolyEthylene Glycol units

PEG25 Bifunctional PEG linker with 25 PolyEthylene Glycol units

PEG1K Bifunctional PEG linker with PolyEthylene Glycol Mol wt of 1000 DaPEG2K Bifunctional PEG linker with PolyEthylene Glycol Mol wt of 2000 DaPEG3.4K Bifunctional PEG linker with PolyEthylene Glycol Mol wt of 3400Da PEG5K Bifunctional PEG linker with PolyEthylene Glycol Mol wt of 5000Da DIG DIGlycolic acid,

IDA β-Ala-Iminodiacetic aicd

Boc-IDA Boc-β-Ala-Iminodiacetic acid

Ac-IDA Ac-β-Ala-Iminodiacetic acid

IDA-Palm Palmityl-β-Ala-Iminodiacetic acid

GTA Glutaric acid

PMA Pemilic acid

AZA Azelaic acid

DDA Dodecanedioic acid

IPA Isopthalic acid

1,3-PDA 1,3-Phenylenediacetic acid

1,4-PDA 1,4-Phenylenediacetic acid

1,2-PDA 1,2-Phenylenediacetic acid

Triazine Amino propyl Triazine di-acid

Boc-Triazine Boc-Triazine di-acid

ADA Amino diacetic acid

AADA n-Acetyl amino acetic acid

PEG4-Biotin PEG4-Biotin (Product number 10199, QuantaBioDesign)

1,4 BMB 1,4-Bis(halo-momethyl)benzene

1,2 BMB 1,2-Bis(halo-momethyl)benzene

1,3 BMB 1,3-Bis(halo-momethyl)benzene,

1,3 BMBip 3,3′-Bis-Halomethyl-Bionenyl

IDA-Biotin N-Biotin-P-Ala-Iminodiacetic acid

2,2 BMBip 2,2′-Bis-Halomethyl-Biphenyl

BMal Bis-Mal-dPEG

The present invention further includes various thioether peptidemonomers or thioether peptide dimers (and subunits thereof) that havebeen substituted with various modified amino acids, including but notlimited to any of those shown in Table 1 or described herein. Forexample, some peptides include Dab, Dap, Pen, Sar, Cit, Cav, HLeu,2-Nal, D-1-Nal, D-2-Nal, Bip, O-Me-Tyr, β-HTrp, β-HPhe, Phe (4-CF3),2-2-Indane, 1-1-Indane, Cyclobutyl, β-HPhe, HLeu, Gla, HPhe, 1-Nal, Nle,homo amino acids, D-amino acids, 3-3-diPhe, cyclobutyl-Ala, HCha,Phe(4-NH2), Bip, β-HPhe, β-Glu, 4-guan, and various N-methylated aminoacids. One having skill in the art will appreciate that additionalsubstitutions may be made to achieve similar desired results, and thatsuch substitutions are within the teaching and spirit of the presentinvention. In certain embodiments, any of the peptides, e.g. peptidedimers and peptide monomer or subunits thereof, described herein orshown in the sequence listing or accompanying figures further comprisesone or more amino acid substitutions, e.g., in certain embodiments, oneor more amino acid residues is substituted with Dab, Dap, Pen, Sar, Cit,Cav, HLeu, 2-Nal, D-1-Nal, D-2-Nal, Bip, O-Me-Tyr, β-HTrp, β-HPhe, Phe(4-CF3), 2-2-Indane, 1-1-Indane, Cyclobutyl, β-HPhe, HLeu, Gla, HPhe,1-Nal, Nle, homo amino acids, D-amino acids, 3-3-diPhe, cyclobutyl-Ala,HCha, Phe(4-NH2), Bip, β-HPhe, β-Glu, 4-guan, or an N-methylated aminoacid, such as, e.g., N-methyl-Arg.

As used herein, “Xaa” can stand for one or more of any naturallyoccurring amino acids, unnatural amino acids, modified amino acids,and/or non-naturally occurring amino acids, including D- and L-aminoacids, aminoacyl residues or any chemical moiety capable of substitutingand amino acid position. In some embodiments, Xaa designates that morethan one amino acid, aminoacyl residue, or chemical residency may occupya given position in the peptide. In particular embodiments, Xaadesignates that a single non-naturally occurring, unnatural, or modifiedamino acid, or an aminoacyl residue or a chemical moiety (e.g., a2-methylbenzoyl moiety) occupies a given position in the polypeptide.

One aspect of the present invention relates to a thioether peptidemonomer, a thioether peptide dimer, or a thioether subunit of a dimermolecule comprising the structure according to Formula (I):

Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Xaa¹²-Xaa¹³-Xaa¹⁴(Formula (I); SEQ ID NO: 388; FIG. 1), or a pharmaceutically acceptablesalt thereof, wherein the peptide monomer or one or both subunits of thethioether peptide dimer comprises a thioether bond between Xaa⁴ andXaa¹⁰ to provide a cyclized structure, and wherein:

Xaa¹ is absent, or selected from the group consisting of any naturallyoccurring amino acid, a suitable isostere, and corresponding D-aminoacids;

Xaa² is absent, or Xaa² is selected from the group consisting of anynaturally occurring amino acid, a suitable isostere, and correspondingD-amino acids;

Xaa³ is absent, or Xaa³ is selected from the group consisting of anynaturally occurring amino acid, a suitable isostere, and correspondingD-amino acids;

Xaa⁴ is an amino acid residue having a side chain with one or twocarbons, and forming a thioether bond with Xaa¹⁰;

Xaa⁵ is selected from the group consisting of N(alpha)-Me-Arg, Arg,HArg, Dap, Dab, Arg-Me-sym, Arg-Me-asym, 4-Guan, Cit, Cav, and suitableisostere replacements;

Xaa⁶ is selected from the group consisting of Ser, Gly, and suitableisostere replacements;

Xaa⁷ is selected from the group consisting of Asp, N-Me-Asp, Asp(OMe),D-Asp, and a suitable isostere replacements;

Xaa⁸ is selected from the group consisting of Thr, Gln, Ser, Asp, Pro,Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu, Val, Tyr, Trp, Leu, Met,and N-Methyl amino acids including N-Me-Thr;

Xaa⁹ is selected from the group consisting of Gln, Asn, Asp, Pro, Gly,Ala, Phe, Leu, Glu, Ile, Val, HLeu, n-Butyl Ala, n-Pentyl Ala, n-HexylAla, Nle, cyclobutyl-Ala, N-Me-Leu, and suitable isostere replacements;

Xaa¹⁰ is selected from the group consisting of Cys, N-Me-Cys, D-Cys,HCys, Pen, and D-Pen;

Xaa¹¹ is absent, or selected from the group consisting of Gly, Gln, Asn,Asp, Ala, Ile, Leu, Val, Met, Thr, Lys, Trp, Tyr, His, Glu, Ser, Arg,Pro, Phe, Sar, 1-Nal, 2-Nal, D-1-Nal, D-2-Nal, HPhe, D-Phe, D-Tyr,Phe(4-F), O-Me-Tyr, dihydro-Trp, Dap, Dab, Dab(Ac), Orn, D-Orn,N-Me-Orn, N-Me-Dap, D-Dap, D-Dab, Bip, Ala(3,3diphenyl), Biphenyl-Ala,aromatic ring substituted Phe, aromatic ring substituted Trp, aromaticring substituted His, hetero aromatic amino acids, N-Me-Lys,N-Me-Lys(Ac), 4-Me-Phe, and corresponding D-amino acids and suitableisostere replacements;

Xaa¹² is absent, or Xaa¹² is selected from the group consisting of Glu,Amide, Lys, COOH, Gln, Pro, Gly, His, Ala, Ile, Phe, Arg, Leu, Val, Tyr,Trp, Met, Gla, Ser, Asn, D-Glu, β-HGlu, 2-Nal, 1-Nal, D-Asp, Bip,β-HPhe, β-Glu, D-Tyr, D-Phe, D-Lys, Dap, Dab, Orn, D-Orn, N-Me-Orn,N-Me-Dap, N-Me-Dab, N-Me Lys, D-Dap, D-Dab, suitable isosteres, andcorresponding D-amino acids;

Xaa¹³ may be absent, or Xaa¹³ is selected from the group consisting ofGln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met,Glu, Ser, Asn, Gla, Dap, Dab, Orn, D-Orn, D-Lys, N-Me-Orn, N-Me-Dap,N-Me-Dab, N-Me-Lys, D-N-Me-Lys, D-Dap, D-Dab, COOH, CONH₂, suitableisosteres, and corresponding D-amino acids;

Xaa¹⁴ is absent, or Xaa¹⁴ is selected from the group consisting of Gln,Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu,Ser, Asn, Gla, Dap, Dab, Orn, D-Orn, D-Lys, N-Me-Orn, N-Me-Dap,N-Me-Dab, N-Me-Lys, D-N-Me-Lys, D-Dap, D-Dab, COOH, CONH₂, suitableisosteres, corresponding D-amino acids, and corresponding N-Methyl aminoacids.

In some embodiments of Formula (I), Xaa⁴ is selected from the groupconsisting of modified Ser, modified HSer, a suitable isostere, andcorresponding D-amino acids and capable of forming a thioether bond withXaa¹⁰. In other instances, Xaa⁴ is an aliphatic acid having from one tofour carbons and capable of forming a thioether bond with Xaa¹⁰. In someinstances, Xaa⁴ is a five- or six-membered alicyclic acid having amodified 2-methyl group that forms a thioether bond with Xaa¹⁰. In someembodiments, Xaa⁴ is acetyl, propionyl, alpha-bromoisobutyryl, or2-methylbenzoyl. In particular embodiments, Xaa⁴ is a 2-methylbenzoylmoiety that forms a thioether bond with Xaa¹⁰.

The present invention also includes peptides comprising the samestructure as shown in Formula (I) or any of the other formulas or tablesdescribed herein, but where the thioether bond is in the reverseorientation. In such embodiments of the invention, it may generally beconsidered that the amino acid residues or other chemical moieties shownat Xaa⁴ are instead present at Xaa¹⁰, and the amino acid residues shownat Xaa¹⁰ are instead present at Xaa⁴, i.e., the amino acid residuecomprising the sulfur of the resulting thioether bond is located at Xaa⁴instead of Xaa¹⁰, and the amino acid residue or other moiety having acarbon side chain capable of forming a thioether bond with Xaa⁴ islocated at Xaa¹⁰. In this reverse orientation, however, the amino acidor chemical moiety at position Xaa¹⁰ is one that comprises a free amine.For example, in particular embodiments, the amino acid at Xaa¹⁰ is aprotected homoserine, such as, e.g., homoserine (OTBDMS). Thus, inparticular reverse orientation embodiments of Formula (I), Xaa¹⁰ is anamino acid residue having a side chain with one or two carbons, andforming a thioether bond with Xaa⁴, and Xaa⁴ is selected from the groupconsisting of Cys, N-Me-Cys, D-Cys, HCys, Pen, and D-Pen. Specificexamples of amino acid residues and other chemical moieties present atcorresponding positions of other formulas and tables are describedherein.

In certain embodiments, a thioether peptide dimer comprises two peptidemonomer subunit of Formula (I), wherein these subunits are linked via alinker moiety through their C- or N-termini. In one embodiment, they arelinked via both their C-termini.

In another aspect, the present invention includes a thioether peptidemolecule (e.g. a peptide monomer, peptide dimer, or a peptide dimersubunit) comprising the structure according to Formula (I-1) (SEQ ID NO:389):

Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Xaa¹²-Xaa¹³-Xaa¹⁴(Formula (I-1)), or a pharmaceutically acceptable salt thereof, whereinthe peptide comprises a thioether bond between Xaa⁴ and Xaa¹⁰, andwherein:

Xaa¹ is absent, or Xaa¹ is any amino acid;

Xaa² is absent, or Xaa² is any amino acid;

Xaa³ is absent, or Xaa³ is any amino acid;

Xaa⁴ is an amino acid, aliphatic acid, alicyclic acid, or modified2-methyl aromatic acid having a side chain with one or two carbons, andcapable of forming a thioether bond with Xaa¹⁰;

Xaa⁵ is selected from the group consisting of N(alpha)-Me-Arg, Arg,HomoArg, Dap, Dab, Arg-Me-sym, Arg-Me-asym, 4-Guan, Cit, Cav, N-Me-Lys,Phe(4-quanidino), Phe(4-carbamoyl amino), Phe(4-NH₂), N-Me-HomoArg, Tyr,His, and suitable isostere replacements;

Xaa⁶ is selected from the group consisting of Ser, Gly, Thr, Ile, andsuitable isostere replacements; wherein if Formula (I-1) is directed toa dimer peptide subunit, then in some embodiments, Xaa⁶ is selected fromthe group consisting of Ser, Gly, Thr, and suitable isosterereplacements;

Xaa⁷ is selected from the group consisting of Asp, N-Me-Asp, Asp(OMe),D-Asp, and suitable isostere replacements;

Xaa⁸ is selected from the group consisting of Thr, Gln, Ser, Asp, Pro,Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu, Val, Tyr, Trp, Leu, Met,HomoLeu, Nle, and N-Methyl amino acids including N-Me-Thr;

Xaa⁹ is selected from the group consisting of Gln, Asn, Asp, Pro, Gly,Ala, Phe, Leu, Glu, Ile, Val, HLeu, n-Butyl Ala, n-Pentyl Ala, n-HexylAla, Nle, cyclobutyl-Ala, Cpa, Aoc, N-Me-Leu, and suitable isosterereplacements;

Xaa¹⁰ is selected from the group consisting of Cys, N-Me-Cys, D-Cys,HCys, Pen, D-Pen, and Pen(=O);

Xaa¹¹ is absent, or Xaa¹¹ is selected from the group consisting of: Trp,Phe, 2-Nal, 1-Nal, Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3), Phe(4-tBu), Bip, Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic,b-homo-Trp, D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl),Phe(4-carbomyl), Phe(3-Carbomyl), Phe (2-carbomyl), Tyr(Me), HomoPhe,N-Me-Phe, N-Me-Tyr, Ser, Sar, Dihydro Trp, Ile, Leu, Ser, Arg, Thr, Sar,and Ser, aromatic amino acids, substituted aromatic amino acids, Gly,Gln, Asn, Asp, Ala, Ile, Leu, Val, Met, Thr, Lys, Trp, Tyr, His, Glu,Ser, Arg, Pro, Phe, Sar, 1-Nal, 2-Nal, D-1-Nal, D-2-Nal, HPhe, D-Phe,D-Tyr, Phe(4-F), O-Me-Tyr, dihydro-Trp, Dap, Dab, Dab(Ac), Orn, D-Orn,N-Me-Orn, N-Me-Dap, D-Dap, D-Dab, Bip, Ala(3,3diphenyl), Biphenyl-Ala,aromatic ring substituted Phe, aromatic ring substituted Trp, aromaticring substituted His, hetero aromatic amino acids, N-Me-Lys,N-Me-Lys(Ac), 4-Me-Phe, Phe(4tBu), Phe(4-OMe), Phe(4-COOH),Phe(2-carbomyl), Phe(3-carbomyl), Phe(CF3), Phe(2,4-diCl),Phe(3,4-diCl), Aic, N-Me-Tyr, N-Me-Phe, Tic, Phe(4CF3), andcorresponding D-amino acids and suitable isostere replacements;

Xaa¹² is absent, or Xaa¹² is selected from the group consisting ofaromatic amino acids, substituted aromatic amino acids, Glu, D-Glu,HomoGlu, Beta-Homo-Glu, Asp, D-HomoGlu, Amide, Lys, COOH, CONH₂, Gln,Pro, Gly, His, Ala, Ile, Phe, Arg, Leu, Val, Tyr, Trp, Met, Gla, Ser,Asn, D-Glu, β-HGlu, 2-Nal, 1-Nal, D-Asp, Bip, β-HPhe, β-Glu, D-Tyr,D-Phe, D-Lys, Dap, Dab, Orn, D-Orn, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-MeLys, D-Dap, D-Dab, D-His, F(4-COOH), Tic, D-Trp, D-Leu, D-Arg, D-Thr,suitable isosteres, and corresponding D-amino acids;

Xaa¹³ is absent, or Xaa¹³ is any amino acid; and

Xaa¹⁴ is absent or any amino acid; wherein in certain embodiments, ifFormula (I-1) is directed to a peptide dimer or subunit thereof, thenXaa¹⁴ is absent or selected from the group consisting of: any amino acidwith an amine side chain, Lys, D-Lys, N-Me-Lys, D-N-Me-Lys, Orn,N-Me-Orn, Dab, N-Me-Dab, Dap, N-Me-Dap, Homo-Lys, D-Dap, D-Dab, D-Orn,Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met,Glu, Ser, Asn, Gla, Cys, HomoCys, COOH, CONH₂, suitable isosteres,corresponding D-amino acids, and corresponding N-Methyl amino acids.

In some embodiments, Xaa⁴ is acetyl, propionyl, alpha-bromoisobutyryl,or 2-methylbenzoyl. In particular embodiments, Xaa⁴ is 2-methylbenzoyl.In particular embodiments, Xaa⁴ is 2-methylbenzoyl.

In certain embodiments, a thioether peptide dimer comprises two peptidemonomer subunit of Formula (I-1), wherein these subunits are linked viaa linker moiety through their C- or N-termini. In one embodiment, theyare linked via both their C-termini.

In particular embodiments, Formula (I-1) is directed to a peptidemonomer or a peptide dimer (or subunit thereof), and Xaa⁷ is selectedfrom the group consisting of Asp, N-Me-Asp, and D-Asp.

In certain embodiments, Xaa¹³ is present and selected from the groupconsisting of Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val,Tyr, Trp, Met, Glu, Ser, Asn, Gla, Dap, Dab, Orn, D-Orn, D-Lys,N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me-Lys, D-N-Me-Lys, D-Dap, D-Dab, COOH,CONH₂, suitable isosteres, and corresponding D-amino acids.

In certain embodiments, Xaa¹⁴ is present. In certain embodiments, Xaa¹⁴is selected from the group consisting of Gln, Pro, Gly, His, Ala, Ile,Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser, Asn, Gla, Dap, Dab,Orn, D-Orn, D-Lys, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me-Lys, D-N-Me-Lys,D-Dap, D-Dab, COOH, CONH₂, suitable isosteres, corresponding D-aminoacids, and corresponding N-Methyl amino acids. In certain embodiments,Xaa¹⁴ is D-Lys, N-Me-Lys, Dap, or Dab. In particular embodiments,Formula (I-1) is directed to a dimer peptide or subunit thereof andXaa¹⁴ is Cys, HomoCys or Pen. In certain embodiments, Xaa¹² and Xaa¹³are absent, and Xaa¹⁴ is D-Lys, N-Me-Lys, Dap, or Dab. In certainembodiments, Xaa¹³ is absent, and Xaa¹⁴ is D-Lys, N-Me-Lys, Dap, or Dab.In some embodiments, Xaa¹², Xaa¹³ and Xaa¹⁴ are absent.

In certain embodiments, the amino acid immediately carboxyl to Xaa¹⁰ isan aromatic amino acid.

In particular embodiments, Formula I-1 is directed to a peptide monomer,dimer, or subunit thereof, and any one or more of Xaa¹, Xaa² or Xaa³ isselected from the group consisting of any naturally occurring aminoacid, a suitable isostere, and corresponding D-amino acids

In particular embodiments, Xaa⁴ is an amino acid residue having a sidechain with one or two carbons.

In particular instances, a peptide monomer, dimer, or subunit thereof ofany of the Formula and peptides described herein comprises Xaa⁴, whereXaa⁴ is selected from the group consisting of modified Ser, modifiedHomoSer (e.g., Homo-Ser-Cl), a suitable isostere, and correspondingD-amino acids. In other instances, Xaa⁴ is an aliphatic acid having fromone to four carbons and forming a thioether bond with Xaa¹⁰. In someinstances, Xaa⁴ is a five- or six-membered alicyclic acid having amodified 2-methyl group that forms a thioether bond with Xaa¹⁰. In someembodiments, Xaa⁴ is a 2-methylbenzoyl moiety.

For some embodiments, at least one of Xaa¹, Xaa², Xaa³, Xaa⁵, Xaa⁷,Xaa⁸, Xaa⁹, Xaa¹¹, Xaa¹², Xaa¹³ and Xaa¹⁴ is N(alpha)Methylated. In someinstances, at least one of Xaa¹, Xaa², Xaa³, Xaa⁴, Xaa¹¹, Xaa¹², Xaa¹³and Xaa¹⁴ are acylated. For example, in some instances one or moreresidues at positions Xaa¹-Xaa⁴, and Xaa¹¹-Xaa¹⁴ are acylated with anacylating organic cisestyl, Hexyl, Palmityl, Lauryl, Oleoyl, and Lauryl,Trifluoromethyl butyric, cyclopentane carboxylic, cyclopropylacetic,4-fluorobenzoic, 4-fluorophenyl acetic, 3-Phenylpropionic,tetrahedro-2H-pyran-4carboxylic, succinic acid, and glutaric acid. Insome instances, small PEG (e.g., PEG4-PEG13) is used as spacer beforeacylations. The present invention also includes reverse order thioetherbond embodiments of Formula (I-1), wherein Xaa¹⁰ is an amino acid,aliphatic acid, alicyclic acid, or modified 2-methyl aromatic acidhaving a side chain with one or two carbons, and capable of forming athioether bond with Xaa⁴; and Xaa⁴ is selected from the group consistingof Cys, N-Me-Cys, D-Cys, HCys, Pen, D-Pen, and Pen(=O). In this reverseorientation, the amino acid or chemical moiety at position Xaa¹⁰ is onethat comprises a free amine. One example of an amino acid or chemicalmoiety that provides a free amine is homoserine or a protectedhomoserine, e.g., homoserine (OTBDMS).

In one aspect, the present invention provides a peptide (e.g. a peptidemonomer, a peptide dimer, or a peptide dimer subunit) comprising thestructure according to Formula (I-2) SEQ ID NO: 34):

Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Xaa¹²-Xaa¹³-Xaa¹⁴(Formula I-2), or a pharmaceutically acceptable salt thereof, whereinthe peptide molecule comprises a thioether bond between Xaa⁴ and Xaa¹⁰,and wherein

Xaa¹ is absent, or Xaa¹ is selected from the group consisting of anynaturally occurring amino acid, a suitable isostere, and correspondingD-amino acids;

Xaa² is absent, or Xaa² is selected from the group consisting of anynaturally occurring amino acid, a suitable isostere, and correspondingD-amino acids;

Xaa³ is absent, or Xaa³ is selected from the group consisting of anynaturally occurring amino acid, a suitable isostere, and correspondingD-amino acids;

Xaa⁴ is an amino acid, aliphatic acid, alicyclic acid, or modified2-methyl aromatic acid having a side chain with one or two carbons, andcapable of forming a thioether bond with Xaa¹⁰;

Xaa⁵ is selected from the group consisting of N(alpha)-Me-Arg, Arg,HomoArg, Dap, Dab, Arg-Me-sym, Arg-Me-asym, 4-Guan, Cit, Cav, N-Me-Lys,Phe (4-quanidino), Phe (4-carbomyl amino), Phe(4-NH2), N-Me-Homo-Arg,Tyr and His, and suitable isostere replacements;

Xaa⁶ is selected from the group consisting of Ser, Gly, Thr, Ile, andsuitable isostere replacements;

Xaa⁷ is selected from the group consisting of Asp, N-Me-Asp, Asp(OMe),D-Asp, and a suitable isostere replacement; wherein in certainembodiments, if Formula (I-2) is directed to a peptide dimer subunitthen Xaa⁷ is selected from the group consisting of Asp, N-Me-Asp, D-Asp,and a suitable isostere replacement;

Xaa⁸ is selected from the group consisting of Thr, Gln, Ser, Asp, Pro,Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu, Val, Tyr, Trp, Leu, Met,hLeu, Nle and N-Methyl amino acids including N-Me-Thr;

Xaa⁹ is selected from the group consisting of Gln, Asn, Asp, Pro, Gly,Ala, Phe, Leu, Glu, Ile, Val, HomoLeu, n-Butyl Ala, n-Pentyl Ala,n-Hexyl Ala, Nle, cyclobutyl-Ala, N-Me-Leu, Cpa, Aoc and suitableisostere replacements; and

Xaa¹⁰ is selected from the group consisting of Cys, N-Me-Cys, D-Cys,HomoCys, Pen, D-Pen, modified HomoSer and modified Ser; wherein incertain embodiments, if Formula (I-2) is directed to a peptide dimersubunit, then Xaa¹⁰ is selected from the group consisting of Cys,N-Me-Cys, D-Cys, HomoCys, Pen, and D-Pen;

Xaa¹¹ is absent, or Xaa¹¹ is selected from the group consisting of orselected from the group consisting of: aromatic amino acids, substitutedaromatic amino acids, Tic, and corresponding D-amino acids and suitableisostere replacements;

Xaa¹² is absent, or Xaa¹² is selected from the group consisting of:aromatic amino acids, substituted aromatic amino acids, Glu, D-Glu,homoGlu, Asp, D-Asp, D-homoGlu, Gla, beta-Homo-Glu, Tic, andcorresponding D-amino acids and suitable isosteres;

Xaa¹³ is absent, or Xaa¹³ is selected from the group consisting of Gln,Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu,Ser, Asn, Gla, Dap, Dab, Orn, D-Orn, D-Lys, N-Me-Orn, N-Me-Dap,N-Me-Dab, N-Me-Lys, D-N-Me-Lys, D-Dap, D-Dab, COOH, CONH₂, suitableisosteres, and corresponding D-amino acids; and

wherein some embodiments, if Formula (I-2) is directed to a peptidemonomer, then Xaa¹⁴ is any amino acid; and

in other embodiments, if Formula (I-2) is directed to a peptide dimersubunit, then Xaa¹⁴ is selected from the group consisting of: any aminoacid with an amine side chain, Lys, D-Lys, N-Me-Lys, D-N-Me-Lys, Orn,N-Me-Orn, Dab, N-Me-Dab, Dap, N-Me-Dap, Homo-Lys, D-Dap, D-Dab, D-Orn,Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met,Glu, Ser, Asn, Gla, Cys, HomoCys, Pen, COOH, CONH₂, suitable isosteres,corresponding D-amino acids, and corresponding N-Methyl amino acids.

The present invention also contemplates reverse order thioether bondembodiments of Formula (I-2), wherein Xaa¹⁰ is an amino acid, aliphaticacid, alicyclic acid, or modified 2-methylbenzoyl moiety acid having afree NH₂ group, and capable of forming a thioether bond with Xaa⁴; andXaa⁴ is selected from the group consisting of Cys, N-Me-Cys, D-Cys,HomoCys, Pen, D-Pen; wherein in certain embodiments, Xaa⁴ is selectedfrom the group consisting of Cys, N-Me-Cys, D-Cys, HomoCys, and Pen.

In one aspect, the present invention provides a peptide (e.g. a peptidemonomer, a peptide dimer, or a peptide dimer subunit) comprising thestructure according to Formula (I-3) SEQ ID NO: 35):

Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Xaa¹²-Xaa¹³-Xaa¹⁴Formula (I-3)), or a pharmaceutically acceptable salt thereof, wherein:

Xaa¹ is absent, Ac, or any amino acid;

Xaa² is absent, Ac, or any amino acid;

Xaa³ is absent, Ac, or any amino acid;

Xaa⁴ is selected from the group consisting of Cys, HomoCys, Pen,Homo-Ser-Cl, Homo-Ser, and a 2-methylbenzoyl moiety;

Xaa⁵ is selected from the group consisting of: N-Me-Arg, Arg, N-Me-Lys,Phe (4-quanidino), Phe(4-carbonylamino), Cit, Phe(4-NH2), N-Me-Homo-Arg,Homo-Arg, Tyr and His;

Xaa⁶ is Ser, Gly, Ile or Thr; wherein in some embodiments, if FormulaI-3 is directed to a peptide monomer then Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is selected from the group consisting of: Thr, Val, Ile, Leu, hLeuand Nle;

Xaa⁹ is selected from the group consisting of: Leu, Nle, Cpa, Cba,HomoLeu, Aoc, and N-Me-Leu;

Xaa¹⁰ is selected from the group consisting of: Cys, D-Cys, HomoCys,Pen, modified HomoSer and modified Ser; wherein in some embodiments, ifFormula I-3 is directed to a peptide monomer, then Xaa¹⁰ is selectedfrom the group consisting of: Cys, D-Cys, HomoCys, and Pen;

Xaa¹¹ is absent or selected from the group consisting of: aromatic aminoacids, and substituted aromatic amino acids;

Xaa¹² is absent or selected from the group consisting of: aromatic aminoacids, substituted aromatic amino acids, Glu, D-Glu, homoGlu, Asp,D-Asp, D-homoGlu, Gla, beta-Homo-Glu, and corresponding D-amino acidsand suitable isosteres;

Xaa¹³ is absent or any amino acid, wherein in particular embodiments,Xaa¹³ is absent or Pro; and

wherein in some embodiments, if Formula I-3 is directed to a peptidemonomer, then Xaa¹⁴ is any amino acid; and

wherein other embodiments, if Formula I-3 is directed to a peptide dimersubunit, then Xaa¹⁴ is absent or selected from the group consisting of:any amino acid with an amine side chain, Lys, D-Lys, N-Me-Lys,D-N-Me-Lys, Orn, N-Me-Orn, Dab, N-Me-Dab, Dap, N-Me-Dap, Homo-Lys,D-Dap, D-Dab, D-Orn, Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu,Val, Tyr, Trp, Met, Glu, Ser, Asn, Gla, Cys, HomoCys, Pen, COOH, CONH₂,suitable isosteres, corresponding D-amino acids, and correspondingN-Methyl amino acids.

The present invention also includes reverse orientation thioether bondembodiments of Formula (I-3), wherein Xaa¹⁰ is selected from the groupconsisting of Homo-Ser-Cl, Homo-Ser, modified Homo-Ser (e.g., HomoSer(OTBDMS)) and a 2-methylbenzoyl moiety with free NH₂ group; and Xaa⁴is selected from the group consisting of: Cys, D-Cys, HomoCys, Pen;wherein in some embodiments, Xaa¹⁰ is selected from the group consistingof: Homo-Ser, modified Homo-Ser and a 2-methylbenzoyl moiety.

In some embodiments of any of the peptides described herein, includingbut not limited to those of Formula (I), (V), (I-1), (I-2), and (I-3),Xaa⁴ is selected from Cys, HomoCys, Pen, and a 2-methylbenzoyl moiety.In certain embodiments, Xaa⁴ is selected from the group consisting of amodified Ser, a modified HomoSer, a suitable isostere, and correspondingD-amino acids. In one embodiment, Xaa⁴ is a Homo-Ser-Cl (before thethioether bond is formed with Xaa¹⁰ whereby the Cl is removed) or aHomoSer precursor (e.g., HomoSer(O-TBDMS). In other instances, Xaa⁴ isan aliphatic acid having from one to four carbons and forming athioether bond with Xaa¹⁰. In some instances, Xaa⁴ is a five- orsix-membered alicyclic acid having a modified 2-methyl group that formsa thioether bond with Xaa¹⁰. In some instances, Xaa⁴ is a2-methylbenzoyl moiety. In some embodiments, the amino acid directlycarboxyl to Xaa¹⁰ is an aromatic amino acid. In some embodiments, Xaa⁷is Asp.

One of skill in the art will appreciate that certain amino acids andother chemical moieties are modified when bound to another molecule. Forexample, an amino acid side chain may be modified when it forms anintramolecular bridge with another amino acid side chain. In addition,when Homo-Ser-Cl binds to an amino acid such as Cys or Pen via athioether bond, the Cl moiety is released. Accordingly, as used herein,reference to an amino acid or modified amino acid, such as Homo-Ser-Cl,present in a peptide dimer of the present invention (e.g., at positionXaa⁴ or position Xaa¹⁰) is meant to include the form of such amino acidor modified amino acid present in the peptide both before and afterforming the intramolecular bond.

In some embodiments of any of the peptides described herein, includingbut not limited to those of Formula (I), (V), (I-1), (I-2), and (I-3),Xaa¹¹ is selected from the group consisting of: Gly, Gln, Asn, Asp, Ala,Ile, Leu, Val, Met, Thr, Lys, Trp, Tyr, His, Glu, Ser, Arg, Pro, Phe,Sar, 1-Nal, 2-Nal, D-1-Nal, D-2-Nal, HPhe, D-Phe, D-Tyr, Phe(4-F),O-Me-Tyr, dihydro-Trp, Dap, Dab, Dab(Ac), Or, D-Orn, N-Me-Orn, N-Me-Dap,D-Dap, D-Dab, Bip, Ala(3,3diphenyl), Biphenyl-Ala, aromatic ringsubstituted Phe, aromatic ring substituted Trp, aromatic ringsubstituted His, hetero aromatic amino acids, N-Me-Lys, N-Me-Lys(Ac),4-Me-Phe, and corresponding D-amino acids and suitable isosterereplacements. In particular embodiments of any of the monomer peptidesdescribed herein, Xaa¹¹ is an aromatic amino acid or a substitutedaromatic amino acid. In certain embodiments, Xaa¹¹ is Phe (4tBu), D-Lys,N-Me-Lys, or D-N-Me-Lys.

In some embodiments of any of the peptides described herein, includingbut not limited to those of Formula (I), (V), (I-1), (I-2), and (I-3),Xaa¹² is selected from the group consisting of Glu, Amide, Lys, COOH,Gln, Pro, Gly, His, Ala, Ile, Phe, Arg, Leu, Val, Tyr, Trp, Met, Gla,Ser, Asn, D-Glu, β-HGlu, 2-Nal, 1-Nal, D-Asp, Bip, β-HPhe, β-Glu, D-Tyr,D-Phe, D-Lys, Dap, Dab, Orn, D-Orn, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-MeLys, D-Dap, D-Dab, suitable isosteres, and corresponding D-amino acids.

In particular embodiments of any of the compounds and genuses describedherein, Xaa⁵ is selected from the group consisting of Cit,Phe(4-carbomyl amino), and N-Me-Homo-Arg; Xaa⁸ is selected from thegroup consisting of Leu, HomoLeu, Nle and Val; Xaa⁹ is selected from thegroup consisting of: Cba, HomoLeu, and Cpa; Xaa¹¹ is selected from thegroup consisting of Tic, Phe(2-carbomyl), Phe(3-carbomyl), Phe (4-COOH),Phe(4-OMe), and Phe(4tBu); Xaa¹² is selected from the group consistingof Aic, Gln, Cit, Glu(OMe), D-His, Tic, Phe(3-COOH), D-Arg, Bip, D-Trp,Phe, D-Phe, D-Val, D-Thr, D-1-Nal, D-2-Nal, Thr, Val; or Xaa¹³ is Pro.

In particular embodiments of any of the peptide described herein,including those of Formula (I), (V), (I-1), (I-2), and (I-3), Xaa⁸ isnot Pro. In particular embodiments of any of the peptide describedherein, including those of Formula (I), (V), (I-1), (I-2), and (I-3),Xaa⁹ is not Pro.

In certain embodiments of any of the peptides (e.g. peptide monomers,peptide dimers or peptide dimer subunits) described herein, includingbut not limited to those of Formula (I), (V), (I-1), (I-2), and (I-3),Xaa¹⁴ is selected from the group consisting of Gln, Pro, Gly, His, Ala,Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser, Asn, Gla, Dap,Dab, Orn, D-Orn, D-Lys, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me-Lys,D-N-Me-Lys, D-Dap, D-Dab, COOH, CONH₂, suitable isosteres, correspondingD-amino acids, and corresponding N-Methyl amino acids. In certainembodiments, Xaa¹⁴ is D-Lys, N-Me-Lys, Dap, or Dab. In some embodimentsof any of the peptide dimer subunits, Xaa¹⁴ (or the C-terminal aminoacid) is Cys, HomoCys or Pen.

In some embodiments of any of the peptides (e.g. peptide monomers,peptide dimers or peptide dimer subunits) described herein, includingbut not limited to those of Formula (I), (V), (I-1), (I-2), and (I-3),Xaa¹⁴ is selected from the group consisting of any amino acid with anamine side chain, Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val,Tyr, Trp, Met, Glu, Ser, Asn, Gla, Dap, Dab, Orn, D-Orn, D-Lys,N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me-Lys, D-N-Me-Lys, D-Dap, D-Dab, COOH,CONH₂, suitable isosteres, corresponding D-amino acids, andcorresponding N-Methyl amino acids

In some embodiments of any of the peptides described herein, includingbut not limited to those of Formula (I), (V), (I-1), (I-2), and (I-3),Xaa¹⁴ is selected from the group consisting of: any amino acid with afree amine side chain, Lys, D-Lys, N-Me-Lys, D-N-Me-Lys, Orn, Dab, Dap,Homo-Lys, D-Dap, D-Dab, or D-Orn.

In some embodiments of any of the peptides (e.g. peptide monomers,peptide dimers or peptide dimer subunits) described herein, includingbut not limited to those of Formula (I), (V), (I-1), (I-2), and (I-3),the amino acid residue directly C-terminal to Xaa¹⁰ is an aromatic aminoacid. In certain embodiments, the amino acid directly C-terminal toXaa¹⁰ is selected from aromatic amino acids, substituted aromatic aminoacids, and Tic. In certain embodiments, the amino acid directlyC-terminal to Xaa¹⁰ is an aromatic amino acid.

In one embodiment of Formula (I-1), herein referred to as Formula (I-A)(SEQ ID NO: 36);

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methyl-benzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is selected from the group consisting of: N-Me-Arg, Arg, N-Me-Lys,Phe (4-quanidino), Phe(4-carbonylamino), Cit, Phe(4-NH2), N-Me-Homo-Arg,Homo-Arg, Tyr and His;

Xaa⁶ is Ser, Gly, Thr or Ile; wherein in some embodiments, if Formula(I-A) is directed to a peptide dimer subunit, then Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is selected from the group consisting of: Thr, Val, Ile, Leu, hLeu,Nle, and Val;

Xaa⁹ is selected from the group consisting of: Leu, Nle, Cpa, Cba,HomoLeu, Aoc, and N-Me-Leu; wherein in some embodiments, if Formula I-Ais directed to a monomer peptide, then Xaa⁹ is selected from the groupconsisting of: Leu, Nle, Cpa, HomoLeu, Aoc, and N-Me-Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys; and

Xaa¹¹ is absent or selected from the group consisting of: Trp, Phe,2-Nal, 1-Nal, Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3), Phe (4-tBu),Bip, Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic,b-homo-Trp, D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl),Phe(4-carbomyl), Phe(3-Carbomyl), Phe (2-carbomyl), Tyr(Me), HomoPhe,N-Me-Phe, N-Me-Tyr, Ser, Sar, Dihydro Trp, Ile, Leu, Arg, Thr, Sar, andSer; wherein in some embodiments, if Formula (I-A) is directed to adimer peptide subunit, then Xaa¹¹ is absent or selected from the groupconsisting of: Trp, Phe, 2-Nal, 1-Nal, Tyr, His, Phe(4-F), Phe(4-CF3),Phe (4-CH3), Phe (4-tBu), Bip, Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3diPhenyl Ala, Tic, b-homo-Trp, D-1-Nal, D-2-Nal, Phe(2,4-diCl),Phe(3,4-diCl), Phe(4-carbomyl), Phe(3-Carbomyl), Phe (2-carbomyl),Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Ser, Sar, Dihydro Trp, Ile, Leu,Arg, and Thr; and

Xaa¹² is absent or selected from the group consisting of: any aromaticamino acid, Glu, D-Glu, homoGlu, Asp, D-Asp, D-homoGlu, D-Asp, Gla,beta-homo-Glu, corresponding D-amino acid, and isosteres; wherein insome embodiments, if Formula (I-A) is directed to a peptide monomer,then Xaa¹² is absent or selected from the group consisting of: anyaromatic amino acid, Glu, D-Glu, homoGlu, Asp, D-Asp, D-homoGlu, Gla,beta-homo-Glu, corresponding D-amino acid, and isosteres;

wherein in some embodiments, if Formula (I-A) is directed to a peptidemonomer, then Xaa¹³ is absent or any amino acid; and

wherein in other embodiments, if Formula (I-A) is directed to a peptidedimer subunit, then Xaa¹³ is absent;

wherein in some embodiments, if Formula (I-A) is directed to a peptidemonomer, then Xaa¹⁴ is any amino acid; and

wherein other embodiments, if Formula (I-A) is directed to a peptidedimer subunit, then Xaa¹⁴ is selected from the group consisting of: anyamino acid with a free amino group on a side chain, Lys, D-Lys,N-Me-Lys, D-N-Me-Lys, Orn, Dab, Dap, Homo-Lys, D-Dap, D-Dab, Cys,HomoCys, Pen, or D-Orn.

In certain embodiments, Formula (I-A) is directed to a peptide monomerand Xaa¹³ is absent.

In one embodiment of Formula (I-1), herein referred to as Formula (I-B)(SEQ ID NO: 37),

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser, Gly, Thr, or Ile; wherein in some embodiments, if Formula(I-B) is directed to a peptide dimer subunit then Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is selected from the group consisting of: Thr, Val, Ile, Leu, hLeuand Nle;

Xaa⁹ is selected from the group consisting of: Leu, Nle, Cpa, Cba,HomoLeu, Aoc, and N-Me-Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3), Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Ser, Sar, DihydroTrp, Ile, Leu, Ser, Arg, Thr, Sar, Ser and any substituted aromaticamino acid and corresponding D-amino acids; wherein in some embodiments,if Formula (I-B) is directed to a peptide dimer subunit, then Xaa¹¹ isselected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal, Tyr, His,Phe(4-F), Phe(4-CF3), Phe (4-CH3), Phe (4-tBu), Bip, Phe(4-COOH), Gly,3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp, D-1-Nal, D-2-Nal,Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl), Phe(3-Carbomyl), Tyr(Me),HomoPhe, N-Me-Phe, N-Me-Tyr, Dihydro Trp, Ile, Leu, Ser, Arg, Thr, Sar,and Ser;

Xaa¹² is selected from the group consisting of: any aromatic amino acid,Glu, D-Glu, homoGlu, Asp, D-Asp, D-homoGlu, Gla, beta-homo-Glu,corresponding D-amino acid and isosteres;

Xaa¹³ is absent;

wherein some embodiments, if Formula (I-B) is directed to a peptidemonomer, then Xaa¹⁴ is any amino acid; and

in other embodiments, if Formula (I-B) is directed to a peptide dimersubunit, then Xaa¹⁴ is selected from the group consisting of: Lys,D-Lys, N-Me-Lys, D-N-Me-Lys, Orn, Dab, Dap, Homo-Lys, D-Dap, D-Dab, Cys,HomoCys, Pen, or D-Orn.

In one embodiment of Formula (I-1), herein referred to as Formula (I-C)(SEQ ID NO: 38),

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser, Gly, Thr, or Ile; wherein in some embodiments, if Formula(I-C) is directed to a peptide dimer subunit, then Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is selected from the group consisting of: Thr, Val, Ile, Leu, hLeuand Nle;

Xaa⁹ is selected from the group consisting of: Leu, Nle, Cpa, Cba,HomoLeu, Aoc, and N-Me-Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3). Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Sar, Dihydro Trp,Ile, Leu, Ser, Arg, Thr, Sar, and Ser; or

Xaa¹² is selected from the group consisting of: any aromatic amino acid,Glu, D-Glu, homoGlu, Asp, D-Asp, D-homoGlu, Gla, beta-homo-Glu,corresponding D-amino acid and isosteres;

Xaa¹³ is absent or any amino acid; wherein in other embodiments, ifFormula (I-C) is directed to a peptide dimer subunit, then Xaa¹³ isabsent; and

wherein in some embodiments, if Formula (I-C) is directed to a peptidemonomer subunit then Xaa¹⁴ is any amino acid; and

wherein in other embodiments, if Formula (I-C) is directed to a peptidedimer subunit then Xaa¹⁴ is selected from the group consisting of: Lys,D-Lys, N-Me-Lys, D-N-Me-Lys, Orn, Dab, Dap, Homo-Lys, D-Dap, D-Dab, Cys,HomoCys, Pen, or D-Orn.

In certain embodiments, Formula (I-C) is directed to a peptide monomerand Xaa¹³ is absent.

In one embodiment of Formula (I-1), herein referred to as Formula (I-D)(SEQ ID NO: 39),

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is Thr or Val;

Xaa⁹ is selected from the group consisting of: Leu, Nle, Cpa, Cba,HomoLeu, Aoc, and N-Me-Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3), Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Ser, Sar, DihydroTrp, Ile, Leu, Ser, Arg, Thr, Sar, and Ser;

Xaa¹² is absent or selected from the group consisting of: any aromaticamino acid, Glu, D-Glu, homoGlu, Asp, D-Asp, D-homoGlu, Gla,beta-homo-Glu, corresponding D-amino acid and isosteres;

Xaa¹³ is absent; and

wherein in some embodiments, if Formula (I-D) is directed to a peptidemonomer then Xaa¹⁴ is any amino acid; and wherein in other embodiments,if Formula (I-D) is directed to a peptide dimer subunit, then Xaa¹⁴ isselected from the group consisting of: Lys, D-Lys, N-Me-Lys, D-N-Me-Lys,Orn, Dab, Dap, Homo-Lys, D-Dap, D-Dab, Cys, HomoCys, Pen, or D-Orn.

In one embodiment of Formula (I-1), herein referred to as Formula (I-E)(SEQ ID NO: 40),

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is Thr or Val;

Xaa⁹ is selected from the group consisting of: Leu, Nle, Cpa, Cba,HomoLeu, Aoc, and N-Me-Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3). Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Ser, Sar, DihydroTrp, Ile, Leu, Ser, Arg, Thr, Sar, and Ser;

Xaa¹² is absent or selected from the group consisting of: any aromaticamino acid, Glu, D-Glu, and beta-homo-Glu;

Xaa¹³ is absent; and,

wherein in some embodiments, if Formula (I-E) is directed to a peptidemonomer, then Xaa¹⁴ is any amino acid; and in other embodiments, ifFormula (I-E) is directed to a peptide dimer subunit, then Xaa¹⁴ isselected from the group consisting of: Lys, D-Lys, N-Me-Lys, D-N-Me-Lys,Orn, Dab, Dap, Homo-Lys, D-Dap, D-Dab, Cys, HomoCys, Pen, or D-Orn.

In one embodiment of Formula (I-1), herein referred to as Formula (I-F)(SEQ ID NO: 41),

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is Thr or Val;

Xaa⁹ is Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3). Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Ser, Sar, DihydroTrp, Ile, Leu, Ser, Arg, Thr, Sar, and Ser;

Xaa¹² is selected from the group consisting of: any aromatic amino acid,Glu, D-Glu, beta-homo-Glu, corresponding D-amino acid and isosteres;

Xaa¹³ is absent; and

wherein in some embodiments, if Formula (I-F) is directed to a peptidemonomer, then Xaa¹⁴ is any amino acid; and wherein in some embodiments,if Formula (I-F) is directed to a peptide dimer subunit, then Xaa¹⁴ isselected from the group consisting of: Lys, D-Lys, N-Me-Lys, D-N-Me-Lys,Orn, Dab, Dap, Homo-Lys, D-Dap, D-Dab, Cys, HomoCys, Pen, or D-Orn.

In certain embodiments, Xaa¹⁴ is selected from the group consisting of:Lys, D-Lys, N-Me-Lys, D-N-Me-Lys.

In one embodiment of Formula (I-1), herein referred to as Formula (I-G)(SEQ ID NO: 42),

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is Thr or Val;

Xaa⁹ is Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3). Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Ser, Sar, DihydroTrp, Ile, Leu, Ser, Arg, Thr, Sar, and Ser;

Xaa¹² is selected from the group consisting of: any aromatic amino acid,Glu, D-Glu, and beta-homo-Glu;

Xaa¹³ is absent; and

wherein in some embodiments, if Formula I-G is directed to a peptidemonomer, then Xaa¹⁴ is any amino acid; and wherein in other embodiments,if Formula I-G is directed to a peptide dimer subunit, then Xaa¹⁴ isselected from the group consisting of: Lys, D-Lys, N-Me-Lys, D-N-Me-Lys,Orn, Dab, Dap, Homo-Lys, D-Dap, D-Dab, Cys, HomoCys, Pen, or D-Orn.

In certain embodiments, Xaa¹⁴ is selected from the group consisting of:D-Lys, N-Me-Lys, and D-N-Me-Lys.

In one embodiment of Formula (I-1), herein referred to as Formula (I-H)(SEQ ID NO: 43),

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser;

Xaa⁷ is Asp;

Xaa⁸ is Thr or Val;

Xaa⁹ is Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3). Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Ser, Sar, DihydroTrp, Ile, Leu, Ser, Arg, Thr, Sar, and Ser;

Xaa¹² is selected from the group consisting of: any aromatic amino acid,Glu, D-Glu, and beta-homo-Glu;

Xaa¹³ is absent; and

wherein in some embodiments, if Formula I-H is directed to a peptidemonomer, then Xaa¹⁴ is any amino acid; and wherein in some embodiments,if Formula I-H is directed to a peptide dimer subunit, then Xaa¹⁴ isselected from the group consisting of: D-Lys, N-Me-Lys, and D-N-Me-Lys.

In one embodiment of Formula (I-1), herein referred to as Formula (I-I)(SEQ ID NO: 44),

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is Thr or Val;

Xaa⁹ is Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3). Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), and HomoPhe;

Xaa¹² is selected from the group consisting of: any aromatic amino acid,Glu, D-Glu, and beta-homo-Glu;

Xaa¹³ is absent; and

wherein in some embodiments, if Formula I-I is directed to a peptidemonomer then Xaa¹⁴ is any amino acid; and wherein in other embodiments,if Formula I-I is directed to a peptide dimer subunit, then Xaa¹⁴ isselected from the group consisting of: D-Lys, N-Me-Lys, and D-N-Me-Lys.

In certain embodiments of Formulas (I), (V), (I-1), (I-2), (I-3), (V),or any of (I-A), (I-B), I-C), (I-D), (I-E), (I-F), (I-G), (I-H), and(I-I), Xaa¹¹ may also be Bpa, Phe(3-Me), Phe(2-Me), Phe(2-CF3), orβ-Me-Phe.

In certain embodiments of Formulas (I), (V), (I-1), (I-2), (I-3), (V) orany of (I-A), (I-B), I-C), (I-D), (I-E), (I-F), (I-G), (I-H), and (I-I),Xaa¹² may also be N-Me-Glu, N-Me-Asp, or alpha-H-Glu.

In particular embodiments of Formulas (I), (V), (I-1), (I-2), (I-3),(V), or any of (I-A), (I-B), I-C), (I-D), (I-E), (I-F), (I-G), (I-H),and (I-I), e.g., when the peptide is a dimer, Xaa¹⁴ is selected from thegroup consisting of: Lys, D-Lys, N-Me-Lys, D-N-Me-Lys, Orn, Dab, Dap,Homo-Lys, D-Dap, D-Dab, Cys, HomoCys, Pen, or D-Orn, while in otherembodiments, Xaa¹⁴ is selected from D-Lys, N-Me-Lys, and D-N-Me-Lys.

In one embodiment of Formula (I-1), Xaa¹ is absent, or Xaa¹ is any aminoacid;

Xaa² is absent, or Xaa² is any amino acid;

Xaa³ is absent, or Xaa³ is any amino acid;

Xaa⁴ is an amino acid, aliphatic acid, alicyclic acid, or modified2-methyl aromatic acid having a side chain with one or two carbons, andcapable of forming a thioether bond with Xaa¹⁰;

Xaa⁵ is selected from the group consisting of N(alpha)-Me-Arg, Arg,HomoArg, Dap, Dab, Arg-Me-sym, Arg-Me-asym, 4-Guan, Cit, Cav, N-Me-Lys,Phe(4-quanidino), Phe(4-carbamoyl amino), Phe(4-NH₂), N-Me-HomoArg, Tyr,His, and suitable isostere replacements;

Xaa⁶ is selected from the group consisting of Ser, Gly, Thr, Ile, andsuitable isostere replacements;

Xaa⁷ is selected from the group consisting of Asp, N-Me-Asp, Asp(OMe),D-Asp, and suitable isostere replacements;

Xaa⁸ is selected from the group consisting of Thr, Gln, Ser, Asp, Pro,Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu, Val, Tyr, Trp, Leu, Met,HomoLeu, Nle, and N-Methyl amino acids including N-Me-Thr;

Xaa⁹ is selected from the group consisting of Gln, Asn, Asp, Pro, Gly,Ala, Phe, Leu, Glu, Ile, Val, HLeu, n-Butyl Ala, n-Pentyl Ala, n-HexylAla, Nle, cyclobutyl-Ala, Cpa, Aoc, N-Me-Leu, and suitable isosterereplacements;

Xaa¹⁰ is selected from the group consisting of Cys, N-Me-Cys, D-Cys,HCys, Pen, D-Pen, and Pen(=O);

Xaa¹¹ is absent or is selected from the group consisting of: Trp, Phe,2-Nal, 1-Nal, Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3), Phe (4-tBu),Bip, Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic,b-homo-Trp, D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl),Phe(4-carbomyl), Phe(3-Carbomyl), Phe (2-carbomyl), Tyr(Me), HomoPhe,N-Me-Phe, N-Me-Tyr, Ser, Sar, Dihydro Trp, Ile, Leu, Ser, Arg, Thr, Sar,and Ser, aromatic amino acids, substituted aromatic amino acids, Gly,Gln, Asn, Asp, Ala, Ile, Leu, Val, Met, Thr, Lys, Trp, Tyr, His, Glu,Ser, Arg, Pro, Phe, Sar, 1-Nal, 2-Nal, D-1-Nal, D-2-Nal, HPhe, D-Phe,D-Tyr, Phe(4-F), O-Me-Tyr, dihydro-Trp, Dap, Dab, Dab(Ac), Orn, D-Orn,N-Me-Orn, N-Me-Dap, D-Dap, D-Dab, Bip, Ala(3,3diphenyl), Biphenyl-Ala,aromatic ring substituted Phe, aromatic ring substituted Trp, aromaticring substituted His, hetero aromatic amino acids, N-Me-Lys,N-Me-Lys(Ac), 4-Me-Phe, Phe(4tBu), Phe(4-OMe), Phe(4-COOH),Phe(2-carbomyl), Phe(3-carbomyl), Phe(CF3), Phe(2,4-diCl),Phe(3,4-diCl), Aic, N-Me-Tyr, N-Me-Phe, Tic, Phe(4CF3), Bpa, Phe(3-Me),Phe(2-Me), Phe(2-CF3), β-Me-Phe, and corresponding D-amino acids andsuitable isostere replacements;

Xaa¹² is absent or selected from the group consisting of aromatic aminoacids, substituted aromatic amino acids, Glu, D-Glu, HomoGlu,Beta-Homo-Glu, Asp, D-HomoGlu, Amide, Lys, COOH, CONH₂, Gln, Pro, Gly,His, Ala, Ile, Phe, Arg, Leu, Val, Tyr, Trp, Met, Gla, Ser, Asn, D-Glu,β-HGlu, 2-Nal, 1-Nal, D-Asp, Bip, β-HPhe, β-Glu, D-Tyr, D-Phe, D-Lys,Dap, Dab, Orn, D-Orn, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me Lys, D-Dap,D-Dab, D-His, F(4-COOH), Tic, D-Trp, D-Leu, D-Arg, D-Thr, N-Me-Glu,N-Me-Asp, alpha-H-Glu, suitable isosteres, and corresponding D-aminoacids;

Xaa¹³ is absent or any amino acid; and

Xaa¹⁴ is absent or any amino acid.

In other embodiments, Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methyl-benzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is selected from the group consisting of: N-Me-Arg, Arg, N-Me-Lys,Phe (4-quanidino), Phe(4-carbonylamino), Cit, Phe(4-NH2), N-Me-Homo-Arg,Homo-Arg, Tyr and His;

Xaa⁶ is Ser, Gly, Thr or Ile;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is selected from the group consisting of: Thr, Val, Ile, Leu, hLeu,Nle, and Val;

Xaa⁹ is selected from the group consisting of: Leu, Nle, Cpa, Cba,HomoLeu, Aoc, and N-Me-Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys; and

Xaa¹¹ is absent or selected from the group consisting of: Trp, Phe,2-Nal, 1-Nal, Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3), Phe (4-tBu),Bip, Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic,b-homo-Trp, D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl),Phe(4-carbomyl), Phe(3-Carbomyl), Phe (2-carbomyl), Tyr(Me), HomoPhe,N-Me-Phe, N-Me-Tyr, Ser, Sar, Dihydro Trp, Ile, Leu, Arg, Thr, Sar, Bpa,Phe(3-Me), Phe(2-Me), Phe(2-CF3), β-Me-Phe, and Ser;

Xaa¹² is absent or selected from the group consisting of: any aromaticamino acid, Glu, D-Glu, homoGlu, Asp, D-Asp, D-homoGlu, D-Asp, Gla,beta-homo-Glu, N-Me-Glu, N-Me-Asp, alpha-H-Glu, corresponding D-aminoacid, and isosteres;

Xaa¹³ is absent or any amino acid; and

Xaa¹⁴ is any amino acid.

In other embodiments,

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser, Gly, Thr, or Ile;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is selected from the group consisting of: Thr, Val, Ile, Leu, hLeuand Nle;

Xaa⁹ is selected from the group consisting of: Leu, Nle, Cpa, Cba,HomoLeu, Aoc, and N-Me-Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3), Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Ser, Sar, DihydroTrp, Ile, Leu, Ser, Arg, Thr, Sar, Bpa, Phe(3-Me), Phe(2-Me),Phe(2-CF3), β-Me-Phe, Ser and any substituted aromatic amino acid andcorresponding D-amino acids;

Xaa¹² is selected from the group consisting of: any aromatic amino acid,Glu, D-Glu, homoGlu, Asp, D-Asp, D-homoGlu, Gla, beta-homo-Glu,N-Me-Glu, N-Me-Asp, alpha-H-Glu, corresponding D-amino acid andisosteres;

Xaa¹³ is absent; and

Xaa¹⁴ is any amino acid.

In other embodiments,

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser, Gly, Thr, or Ile;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is selected from the group consisting of: Thr, Val, Ile, Leu, hLeuand Nle;

Xaa⁹ is selected from the group consisting of: Leu, Nle, Cpa, Cba,HomoLeu, Aoc, and N-Me-Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3). Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Sar, Dihydro Trp,Ile, Leu, Ser, Arg, Thr, Sar, Bpa, Phe(3-Me), Phe(2-Me), Phe(2-CF3),β-Me-Phe, and Ser;

Xaa¹² is selected from the group consisting of: any aromatic amino acid,Glu, D-Glu, homoGlu, Asp, D-Asp, D-homoGlu, Gla, beta-homo-Glu,N-Me-Glu, N-Me-Asp, alpha-H-Glu, corresponding D-amino acid andisosteres;

Xaa¹³ is absent or any amino acid; and

Xaa¹⁴ is any amino acid.

In other embodiments:

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is Thr or Val;

Xaa⁹ is selected from the group consisting of: Leu, Nle, Cpa, Cba,HomoLeu, Aoc, and N-Me-Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3), Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Ser, Sar, DihydroTrp, Ile, Leu, Ser, Arg, Thr, Sar, Bpa, Phe(3-Me), Phe(2-Me),Phe(2-CF3), β-Me-Phe, and Ser;

Xaa¹² is absent or selected from the group consisting of: any aromaticamino acid, Glu, D-Glu, homoGlu, Asp, D-Asp, D-homoGlu, Gla,beta-homo-Glu, N-Me-Glu, N-Me-Asp, alpha-H-Glu, corresponding D-aminoacid and isosteres;

Xaa¹³ is absent; and

Xaa¹⁴ is any amino acid.

In other embodiments:

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is Thr or Val;

Xaa⁹ is selected from the group consisting of: Leu, Nle, Cpa, Cba,HomoLeu, Aoc, and N-Me-Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3). Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Ser, Sar, DihydroTrp, Ile, Leu, Ser, Arg, Thr, Sar, Bpa, Phe(3-Me), Phe(2-Me),Phe(2-CF3), β-Me-Phe, and Ser;

Xaa¹² is absent or selected from the group consisting of: any aromaticamino acid, Glu, D-Glu, beta-homo-Glu, N-Me-Glu, N-Me-Asp, alpha-H-Glu;

Xaa¹³ is absent; and

Xaa¹⁴ is any amino acid.

In other embodiments:

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is Thr or Val;

Xaa⁹ is Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3). Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Ser, Sar, DihydroTrp, Ile, Leu, Ser, Arg, Thr, Sar, Bpa, Phe(3-Me), Phe(2-Me),Phe(2-CF3), β-Me-Phe, and Ser;

Xaa¹² is selected from the group consisting of: any aromatic amino acid,Glu, D-Glu, beta-homo-Glu, N-Me-Glu, N-Me-Asp, alpha-H-Glu,corresponding D-amino acid and isosteres;

Xaa¹¹ is absent; and

Xaa¹⁴ is any amino acid.

In other embodiments:

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is Thr or Val;

Xaa⁹ is Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3). Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Ser, Sar, DihydroTrp, Ile, Leu, Ser, Arg, Thr, Sar, Bpa, Phe(3-Me), Phe(2-Me),Phe(2-CF3), β-Me-Phe, and Ser;

Xaa¹² is selected from the group consisting of: any aromatic amino acid,Glu, D-Glu, N-Me-Glu, N-Me-Asp, alpha-H-Glu, and beta-homo-Glu;

Xaa¹¹ is absent; and

Xaa¹⁴ is any amino acid.

In other embodiments:

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser;

Xaa⁷ is Asp;

Xaa⁸ is Thr or Val;

Xaa⁹ is Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3). Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), HomoPhe, N-Me-Phe, N-Me-Tyr, Ser, Sar, DihydroTrp, Ile, Leu, Ser, Arg, Thr, Sar, Bpa, Phe(3-Me), Phe(2-Me),Phe(2-CF3), β-Me-Phe, and Ser;

Xaa¹² is selected from the group consisting of: any aromatic amino acid,Glu, D-Glu, N-Me-Glu, N-Me-Asp, alpha-H-Glu, and beta-homo-Glu;

Xaa¹³ is absent; and

Xaa¹⁴ is any amino acid.

In other embodiments:

Xaa¹ is absent or any amino acid;

Xaa² is absent or any amino acid;

Xaa³ is absent or any amino acid;

Xaa⁴ is a 2-methylbenzoyl moiety or a modified HomoSer, optionallyHomo-Ser-Cl;

Xaa⁵ is N-Me-Arg;

Xaa⁶ is Ser;

Xaa⁷ is Asp or D-Asp;

Xaa⁸ is Thr or Val;

Xaa⁹ is Leu;

Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys;

Xaa¹¹ is selected from the group consisting of: Trp, Phe, 2-Nal, 1-Nal,Tyr, His, Phe(4-F), Phe(4-CF3), Phe (4-CH3). Phe (4-tBu), Bip,Phe(4-COOH), Gly, 3,3-DiPhenylGly, 3,3 diPhenyl Ala, Tic, b-homo-Trp,D-1-Nal, D-2-Nal, Phe(2,4-diCl), Phe(3,4-diCl), Phe(4-carbomyl),Phe(3-Carbomyl), Tyr(Me), Bpa, Phe(3-Me), Phe(2-Me), Phe(2-CF3),β-Me-Phe, and HomoPhe;

Xaa¹² is selected from the group consisting of: any aromatic amino acid,Glu, D-Glu, N-Me-Glu, N-Me-Asp, alpha-H-Glu, and beta-homo-Glu;

Xaa¹³ is absent; and

Xaa¹⁴ is any amino acid.

In some embodiments of any of the peptides (e.g. peptide monomers, orpeptide dimers or subunits thereof) described herein, including but notlimited to those of Formula (I) (including (I-A)-(I-I), (I-1), (I-2) and(I-3)) or Formula (V), Xaa⁷ is Asp.

In some embodiments of any of the peptides (e.g. peptide monomers, orpeptide dimers or subunits thereof) described herein, including but notlimited to those of Formula (I) (including (I-A)-(I-I), (I-1), (I-2) and(I-3) or Formula (V)), the N-terminus of the peptide is acylated.

In some embodiments of any of the peptides (e.g. peptide monomers orpeptide dimers or subunits thereof) described herein, including but notlimited to those of Formula (I) (including (I-A)-(I-I), (I-1), (I-2) and(I-3) or Formula (V)), Xaa¹⁴ or the C-terminal amino acid does notcomprise a free amine.

In some embodiments of any of the peptides (e.g. peptide monomers orpeptide dimers or subunits thereof) described herein, including but notlimited to those of Formula (I) (including (I-A)-(I-I), (I-1), (I-2) and(I-3) or Formula (V)), Xaa¹⁴ or the C-terminus comprises an NH₂ or anOH. In particular embodiments, Xaa¹³ is D-Lys Xaa¹⁴ or the C-terminus isan OH.

In some embodiments of any of the peptide (e.g. peptide monomers orpeptide dimers or subunits thereof) described herein, including but notlimited to those of Formula (I) (including (I-A)-(I-I), (I-1), (I-2) and(I-3) or Formula (V)), a free amine in the C-terminal amino acid of thepeptide monomer is capped, e.g., with an acetyl group.

In some embodiments of any of the peptides (e.g. peptide monomers orpeptide dimers or subunits thereof) described herein, including but notlimited to those of Formula (I) (including (I-A), (I-I), (I-1), (I-2)and (I-3)) or Formula (V), the peptide monomer or dimer subunitcomprises an intramolecular thioether bond between Xaa⁴ and Xaa¹⁰. Incertain embodiments, Xaa⁴ is a 2-methylbenzoyl moiety, and Xaa¹⁰ is Pen.In certain embodiments, Xaa⁴ is Homo-Ser-Cl, and Xaa¹⁰ is Cys, D-Cys, orHomoCys.

In some embodiments of any of the peptides (e.g. peptide monomers orpeptide dimers or subunits thereof) described herein, including but notlimited to those of Formula (I) (including (I-A)-(I-I), (I-1), (I-2) and(I-3)) or Formula (V), at least one of Xaa¹, Xaa², Xaa³, Xaa⁵, Xaa⁷,Xaa⁸, Xaa⁹, Xaa¹¹, Xaa¹², Xaa¹³ and Xaa¹⁴ is N(alpha)Methylated.

In some instances of any of the peptides (e.g. peptide monomers orpeptide dimers or subunits thereof) described herein, any of Xaa¹-Xaa⁴,and Xaa¹¹-Xaa¹⁴ are acylated. For example, in some instances one or moreresidues at positions Xaa¹-Xaa⁴, and Xaa¹¹-Xaa¹⁴ are acylated with anacylating organic compound selected from the group consisting of2-me-Trifluorobutyl, Trifluoropentyl, Acetyl, Octonyl, Butyl, Pentyl,Hexyl, Palmityl, Lauryl, Oleoyl, and Lauryl, Trifluoromethyl butyric,cyclopentane carboxylic, cyclopropylacetic, 4-fluorobenzoic,4-fluorophenyl acetic, 3-Phenylpropionic,tetrahedro-2H-pyran-4carboxylic, succinic acid, and glutaric acid. Insome instances, small PEG (e.g., PEG4-PEG13) is used as spacer beforeacylations.

In certain embodiments, the N-terminus of a peptide monomer or peptidedimer subunit represented by Formula (I) (including (I-A)-(I-I), (I-1),(I-2) and (I-3)), or Formula (II) or Formula (V) or Formula (VI), or anyother peptide described herein, can be modified by one to three suitablegroups, as represented by Xaa¹, Xaa², and Xaa³ in Formula (I), (I-A),(I-B) and (I-C) or Formula (V). The N-terminus may further be acylatede.g., as described herein with respect to peptide monomers or peptidedimer subunits of Formula (I), Formula (V), Formula (II), and Formula(VI). In some instances, the N-terminus further comprises a suitablelinker moiety or other modifying group.

Similarly, in certain embodiments, the C-terminus of a peptide monomeror dimer subunit represented by Formula (I) (including (I-A)-(I-I)),(I-1), (I-2) and (I-3), or Formula (V), or a peptide monomer or peptidedimer subunit of Formula (II), or any other peptide described herein,can be modified by a suitable group. For example, the C-terminus may beacylated. In some instances, the C-terminus further comprises a suitablelinker moiety or modifying group, as disclosed herein. In certainembodiments, the C-terminus comprises NH₂ or OH.

In some embodiments, Xaa¹, Xaa², and Xaa³ of Formula (I) (including(I-1)-(I-I)), (I-1), (I-2) and (I-3) or Formula (V) are absent. Inparticular embodiments Xaa¹, Xaa², and Xaa³ of any peptide dimer subunitdescribed herein are absent. In other embodiments, Xaa¹ is absent, andXaa² and Xaa³ represent suitable groups for modifying the N-terminus ofthe peptide monomer or peptide dimer subunit. Further, in someembodiments Xaa¹ and Xaa² are absent, and Xaa³ represents a singlesuitable group for modifying the N-terminus of the peptide monomer orpeptide dimer subunit.

With continued reference to the peptide monomers and peptide of thegeneral formula of Formula (I), (I-1), (I-2) and (I-3) or Formula (V),Xaa¹⁻³ may comprise any naturally occurring amino acid, a suitableisostere, or corresponding D-amino acid. In some embodiments, at leastone of Xaa¹⁻³ is absent. For example, in some instances Xaa¹ is absent,whereby Xaa² is the N-terminus. In other instances Xaa¹ and Xaa² areabsent, whereby Xaa³ is the N-terminus. Further still, in some instancesXaa¹⁻³ are absent, whereby Xaa⁴ is the N-terminus. In some embodiments,the N-terminal residue is acylated or comprises a free amine. In someembodiments, the N-terminal residue of the peptide monomer or peptidedimer subunit is a 2-methyl benzoyl moiety (abbreviated herein as2-benzyl).

In certain embodiments, peptide monomers, or peptide dimers havingsubunits of Formula (I) (including (I-A)-(I-I)), (I-1), (I-2) and (I-3)or Formula (V), or any other peptide described herein, the amino acidresidue directly C-terminal to Xaa¹⁰ is an aromatic amino acid.

In other embodiments, the N-terminal residue of peptide monomers orpeptide dimer subunits of Formula (I) (including (I-A)-(I-I), (I-1),(I-2) and (I-3)), or any other peptide described herein, furthercomprises a suitable linker moiety, e.g., a linker moiety, or modifyinggroup selected from the group consisting of DIG, PEG4, PEG13, PEG25,PEG1K, PEG2K, PEG4K, PEG5K, Polyethylene glycol having molecular weightfrom 400 Da to 40,000 Da, PEG having a molecular weight of 40,000 Da to80,000 Da, IDA, Ac-IDA, ADA, Glutaric acid, AADA, suitable aliphaticacids, suitable aromatic acids, and heteroaromatic acids.

In various embodiments of any of the peptides (e.g. peptide monomers,peptide dimers, or subunits thereof) described herein, one or more ofthe amino acids represented by Xaa¹⁻³ may be either absent or selectedfrom the group consisting of any naturally occurring amino acid, asuitable isostere, and corresponding D-amino acids. When Xaa¹ and Xaa²are absent, Xaa³ is the N-terminus. When Xaa¹⁻³ are absent, Xaa⁴ is theN-terminus.

In some embodiments, Xaa⁴ is an amino acid residue having a side chainwith one or two carbons, and forming a thioether bond with Xaa¹⁰. Insome instances, Xaa⁴ is selected from the group consisting of modifiedSer, modified HSer, a suitable isostere, and corresponding D-aminoacids. In other instances, Xaa⁴ is an aliphatic acid having from one tofour carbons and forming a thioether bond with Xaa¹⁰. In some instances,Xaa⁴ is a five- or six-membered alicyclic acid having a modified2-methyl group that forms a thioether bond with Xaa¹⁰. In someembodiments, Xaa⁴ is a 2-methyl-benzoyl moiety or a modified formthereof. In certain embodiments, Xaa⁴ Cys, Pen, homocys, D-Pen, D-Cys orD-homocys. In certain embodiments, Xaa⁴ is 2-chloromethylbenzoic acid,2-chloro-acetic acid, 3-chloro-propanoic acid, 4-chloro-butyric acid,3-chloro-isobutyric acid, Ser(Cl); Xaa¹⁰ is Cys, Pen, D-Cys, HomoCys;and the intramolecular bond is a thioether bond. One of skill in the artwill appreciate that upon bonding with another amino acid, e.g., Xaa¹⁰,the Cl of hSer(Cl) will be removed.

For each embodiment of the peptide monomers or peptide dimer subunits ofFormula (I) and (I-A) or Formula (V), and any of the peptide monomers orpeptide dimers described herein, a thioether bond exists between Xaa⁴and Xaa¹⁰ in the monomer peptides or in one or both of the peptide dimersubunits. Thus, the thioether peptide monomers or peptide dimer subunitsof the present invention are cyclized through a thioether bond.

In some embodiments of any of the peptides described herein, Xaa⁵ isselected from the group consisting of N(alpha)-Me-Arg, Arg, HArg, Dap,Dab, Arg-Me-sym, Arg-Me-asym, 4-Guan, Cit, Cav, and suitable isosterereplacements. In some embodiments, Xaa⁵ is N(alpha)Methylated.Preferably, Xaa⁵ is N-Me-Arg. In other embodiments, preferably Xaa⁵ isArg.

In some embodiments of any of the peptides (e.g. peptide monomers,peptide dimers, or subunits thereof), described herein, Xaa⁶ is selectedfrom the group consisting of Ser, Gly, Thr, Ile, and suitable isosterereplacements. Preferably, Xaa⁶ is Ser. In some embodiments of any of thepeptide dimer subunits described herein, Xaa⁶ is selected from the groupconsisting of Ser, Gly, Thr, Ile, and suitable isostere replacements. Insome embodiments of any of the peptide monomers described herein, Xaa⁶is selected from the group consisting of Ser, Gly, and suitable isosterereplacements.

In some embodiments of any of the peptide monomers or dimers describedherein, Xaa⁷ is selected from the group consisting of Asp, N-Me-Asp,D-Asp, Asp(OMe), and a suitable isostere replacements. In someembodiments of any of the peptide dimers described herein, Xaa⁷ isselected from the group consisting of Asp, N-Me-Asp, D-Asp, and asuitable isostere replacements. In some embodiments, Xaa⁷ isN(alpha)Methylated. Preferably, Xaa⁷ is Asp.

In some embodiments of any of the peptides described herein, Xaa⁸ isselected from the group consisting of Thr, Gln, Ser, Asp, Pro, Gly, His,Ala, Ile, Phe, Lys, Arg, Asn, Glu, Val, Tyr, Trp, Leu, Met, and N-Methylamino acids including N-Me-Thr, and suitable isostere replacements. Insome embodiments, Xaa⁸ is N(alpha)Methylated. Preferably, Xaa⁸ is Thr.

In some embodiments of any of the peptides described herein, Xaa⁹ isselected from the group consisting of Gln, Asn, Asp, Pro, Gly, Ala, Phe,Leu, Glu, Ile, Val, HLeu, n-Butyl Ala, n-Pentyl Ala, n-Hexyl Ala, Nle,cyclobutyl-Ala, N-Me-Leu, and suitable isostere replacements. In someembodiments, Xaa⁹ is N(alpha)Methylated. In certain embodiments, Xaa⁹ isLeu.

In some embodiments of any of the peptide monomers or peptide dimersubunits described herein, Xaa¹⁰ is selected from the group consistingof Cys, N-Me-Cys, D-Cys, HCys, Pen, and D-Pen. In some embodiments,Xaa¹⁰ is selected from the group consisting of Cys, N-Me-Cys, D-Cys,HCys, and Pen. In one embodiment, Xaa¹⁰ is Pen. In another embodiment,Xaa¹⁰ is preferably Cys.

In some embodiments of any of the peptides described herein, Xaa¹¹ isabsent, or Xaa¹¹ is selected from the group consisting of Gly, Gln, Asn,Asp, Ala, Ile, Leu, Val, Met, Thr, Lys, Trp, Tyr, His, Glu, Ser, Arg,Pro, Phe, Sar, 1-Nal, 2-Nal, D-1-Nal, D-2-Nal, HPhe, Phe(4-F), O-Me-Tyr,dihydro-Trp, D-Phe, D-Tyr, Dap, Dab, Dab(Ac), Orn, D-Orn, N-Me-Orn,N-Me-Dap, D-Dap, D-Dab, Bip, Ala(3,3diphenyl), Biphenyl-Ala, aromaticring substituted Phe, aromatic ring substituted Trp, aromatic ringsubstituted His, hetero aromatic amino acids, N-Me-Lys, N-Me-Lys(Ac),4-Me-Phe, and corresponding D-amino acids and suitable isosterereplacements. In some embodiments, Xaa¹¹ is preferably Trp. In someother embodiments, Xaa¹¹ is Phe. In some embodiments, Xaa¹¹ is F(4tBu),F(4-COOH), Bip, 1-Nal or 2-Nal. In particular embodiments of peptidemonomers described herein, Xaa¹¹ is N(alpha)Methylated. In certainembodiments of peptide monomers or peptide dimer subunits describedherein, Xaa¹¹ is Phe. In some embodiments, Xaa¹¹ is N(alpha)Methylated.Further, in some embodiments Xaa¹¹ is acylated.

In at least one embodiment of peptide monomers or peptide dimer subunitsdescribed herein, Xaa¹¹ is absent and Xaa¹⁰ is the C-terminus. WhenXaa¹²⁻¹⁴ are absent, Xaa¹¹ is the C-terminus of the subunit. When Xaa¹¹is the C-terminus of the subunit, Xaa¹¹ may be modified to include asuitable linker moiety in accordance with the present invention.

In some embodiments of peptide monomers or peptide dimers describedherein, Xaa¹² is absent, or Xaa¹² is selected from the group consistingof Glu, Lys, COOH, CONH₂, Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg,Leu, Val, Tyr, Trp, Met, Gla, Ser, Asn, D-Glu, β-HGlu, 2-Nal, 1-Nal,D-Asp, Bip, β-HPhe, β-Glu, D-Tyr, D-Phe, D-Lys, Dap, Dab, Orn, D-Orn,N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me Lys, D-Dap, D-Dab, suitableisosteres, and corresponding D-amino acids. In some embodiments ofpeptide dimers described herein, Xaa¹² is absent, or Xaa¹² is selectedfrom the group consisting of Glu, Lys, Gln, Pro, Gly, His, Ala, Ile,Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Gla, Ser, Asn, D-Glu, β-HGlu,2-Nal, 1-Nal, D-Asp, Bip, β-HPhe, β-Glu, D-Tyr, D-Phe, D-Lys, Dap, Dab,Orn, D-Orn, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me Lys, D-Dap, D-Dab,suitable isosteres, and corresponding D-amino acids. In certainembodiments, Xaa¹² is Glu, D-Glu, β-HGlu, or Asp. In some embodiments,Xaa¹² is β-Hglu.

In some embodiments of the peptide monomer or peptide dimers describedherein, Xaa¹³ and Xaa¹⁴ are absent, and Xaa¹² is the C-terminus of thesubunit. In some embodiments of the peptide dimers described herein,when Xaa¹² is the C-terminus of the subunit, Xaa¹² may be modified toinclude a suitable linker moiety in accordance with the presentinvention.

In some embodiments of any of the peptides (e.g. peptide monomers,peptide dimers, or subunits thereof) described herein, Xaa¹³ is absent,or Xaa¹³ is selected from the group consisting of Gln, Pro, Gly, His,Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser, Asn, Gla,Dap, Dab, Orn, D-Orn, D-Lys, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me-Lys,D-N-Me-Lys, D-Dap, D-Dab, suitable isosteres, and corresponding D-aminoacids. In some embodiments of peptide monomers described herein, Xaa¹³is absent, or Xaa¹³ is selected from COOH and CONH₂. In at least oneembodiment, Xaa¹³ is Lys. Further still in some embodiments Xaa¹³ isD-Lys. In some embodiments of the peptide dimer subunits describedherein, when Xaa¹⁴ is absent, Xaa¹³ is the C-terminus; and when Xaa¹³ isthe C-terminus of the subunit, Xaa¹³ may be modified to include asuitable linker moiety in accordance with the present invention.

Further, in some embodiments of the peptide monomers or dimer subunitsdescribed herein, Xaa¹⁴ is absent, or Xaa¹⁴ is selected from the groupconsisting of Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val,Tyr, Trp, Met, Glu, Ser, Asn, Gla, Dap, Dab, Orn, D-Orn, D-Lys,N-Me-Orn, N-Me-Dap, N-Me-Dab, COOH, CONH₂, N-Me-Lys, D-N-Me-Lys, D-Dap,D-Dab, suitable isosteres, corresponding D-amino acids, andcorresponding N-Methyl amino acids. Further, in some embodiments of thepeptide dimer subunits described herein, Xaa¹⁴ is absent, or Xaa¹⁴ isselected from the group consisting of Gln, Pro, Gly, His, Ala, Ile, Phe,Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser, Asn, Gla, Dap, Dab, Orn,D-Orn, D-Lys, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me-Lys, D-N-Me-Lys, D-Dap,D-Dab, suitable isosteres, corresponding D-amino acids, andcorresponding N-Methyl amino acids. In at least one embodiment of thepeptide monomers and dimer subunits described herein, Xaa¹⁴ is Lys,D-Lys, or N-Me-Lys. In some embodiments of the peptide monomer orpeptide dimer subunits of the present invention, Xaa¹⁴ is Cys, HomoCysor Pen. In some embodiments of the peptide monomer or peptide dimersubunits of the present invention, Xaa¹⁴ is Cys, D-Cys, HomoCys, Pen, orD-Pen.

In some embodiments of any of the peptide monomers or dimer subunitsdescribed herein, Xaa¹² is present, Xaa¹³ is absent, and Xaa¹⁴ ispresent. In particular embodiments, Xaa¹¹ is Phe(4tBu), Phe(4-COOH),Bip, 2-Nal or 1-Nal; Xaa¹² is Glu or β-homoGlu, Xaa¹³ is absent, andXaa¹⁴ is D-Lys or N-Me-Lys.

In at least one embodiment of the dimer subunits described herein, Xaa¹⁴is the C-terminus, and when Xaa¹⁴ is the C-terminus of the subunit,Xaa¹⁴ may be modified to include a linker moiety in accordance with thepresent invention.

In at least one embodiment of peptide monomers and peptide dimersubunits, including peptide monomers and dimers of Formula (I),described herein, Xaa¹¹⁻¹⁴ are absent, whereby Xaa¹⁰ is the C-terminus.When Xaa¹²⁻¹⁴ are absent, Xaa¹¹ is the C-terminus. Similarly, when Xaa¹³and Xaa¹⁴ are absent, Xaa¹² is the C-terminus. Further, when Xaa¹⁴ isabsent, Xaa¹³ is the C-terminus. In some embodiments, the C-terminus ofthe thioether peptide monomer or dimer subunit is modified to include asuitable linker moiety (e.g. a linker moiety) or modifying group inaccordance with the present invention.

In certain embodiments of any of the peptide monomers or dimer subunits(e.g. the peptide monomers and dimers of Formula (I)) described herein,Xaa¹, Xaa² and Xaa³ are absent, and the N-terminus of the peptidecomprises an aromatic group that is capable of forming a thioether bondwith Xaa¹⁰. In some embodiments, Xaa⁴ comprises a 2-methylbenzoyl moietyforming an amide bond with Xaa⁵, and further comprising a methyl groupforming a thioether bond with Xaa¹⁰. The 2-methylbenzoyl moiety furthercomprises substituent R-groups represented by R1-R4, e.g., as shown inFIG. 4.

In some instances of peptide monomers or dimers described herein, atleast one substituent R-group of Xaa¹ is a free amine, whereby theN-terminus of the thioether monomer or dimer peptide of, e.g., Formula(I) or Formula (I-1), may be extended. In other instances, one or moresubstituent groups represented by R1-R4 is selected from the groupconsisting of hydrogen, a methyl group, a fluorocarbon group, ahydrocarbon, Cl, CF3, OMe, OEt, CONH₂, an aromatic group, a smallpegylation group, a terminal modifying group, an acylation, a freeamine, and an acid. In some embodiments, one or more substituent groupsrepresented by R1-R4 is selected from the group consisting of hydrogen,a methyl group, a fluorocarbon group, a hydrocarbon, Cl, CF3, OMe, OEt,CONH₂, CH3, CH2CH3, an aromatic group, a small pegylation group, aterminal modifying group, an acylation, a free amine, and an acid.

In particular embodiments of any of the peptides herein, including thosecomprising a structure of any one of Formulas (I), (I-1), (I-2), (I-3),(V) or (I-A)-(I-I) or Formula (V), the thioether bond is in the reverseorder, such that the amino acid residues and chemical moieties shown inXaa⁴ are instead present in Xaa¹⁰, and the amino acid resides shown atXaa¹⁰ are instead present at Xaa⁴. In this reverse orientation, theamino acid or chemical moiety at position Xaa¹⁰ is one that comprises afree amine.

In some embodiments of the peptide monomers and dimer subunits describedherein, the C-terminal residue of Formula (I) or Formula (V) or anypeptide monomer or peptide dimer described herein further comprises amodifying group or a suitable linker moiety, e.g., a modifying group orlinker selected from the group consisting of DIG, PEG4, PEG13, PEG25,PEG1K, PEG2K, PEG4K, PEG5K, Polyethylene glycol having molecular weightfrom 400 Da to 40,000 Da, PEG having a molecular weight of 40,000 Da to80,000 Da, IDA, Ac-IDA, ADA, Glutaric acid, Succinic acid, Isophthalicacid, 1,3-phenylenediacetic acid, 1,4-phenylenediacetic acid,1,2-phenylenediacetic acid, AADA, suitable aliphatic acids, suitablearomatic acids, heteroaromatic acids. Examples of other linkers aredescribed herein and include but are not limited to those linkers shownin Table 2.

Referring now to FIG. 4, one aspect of the present invention relates toa thioether peptide monomer or dimer (or subunit of a peptide dimermolecule) comprising the structure according to Formula (II):

-   -   Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹ (SEQ ID        NO: 2), or a pharmaceutically acceptable salt thereof, wherein        the peptide monomer or each subunit of the thioether peptide        dimer comprises a thioether bond between Xaa¹ and Xaa⁷.

The N-terminus of a peptide monomer or dimer subunit represented byFormula (II) comprises an aromatic group that is capable of forming athioether bond with Xaa⁷. In some embodiments, Xaa¹ comprises a2-methylbenzoyl moiety forming an amide bond with Xaa², and furthercomprising a methyl group forming a thioether bond with Xaa⁷. The2-methylbenzoyl moiety may further comprise substituent R-groupsrepresented by R1-R4, e.g., as shown in FIG. 4, including thosedescribed herein.

In some instances, at least one substituent R-group of Xaa¹ is a freeamine, whereby the N-terminus of the thioether peptide of Formula (II)may be extended. In other instances, one or more substituent groupsrepresented by R1-R4 is selected from the group consisting of hydrogen,a methyl group, a fluorocarbon group, a hydrocarbon, Cl, CF3, OMe, OEt,CONH₂, an aromatic group, a small pegylation group, a terminal modifyinggroup, an acylation, a free amine, and an acid.

For each embodiment of Formula (II) or Formula (VI), a thioether bondexists between Xaa¹ and Xaa⁷. Thus, the thioether peptide monomers anddimer subunits of the present invention are cyclized through a thioetherbond. In one embodiment, Xaa⁷ is Cys. In another embodiment, preferablyXaa⁷ is Pen. In other embodiments, Xaa⁷ is D-Cys or homo-Cys.

In some embodiments of peptides (e.g. peptide monomers, dimers, or dimersubunits) described herein, Xaa¹ comprises an R group that is capable ofbeing acylated via an acylating organic compound. In other instances,Xaa¹ of a peptide dimer subunit comprises an R group that is capable ofbeing modified with a suitable linker moiety, whereby the N-terminusesof two peptide dimer subunits according to Formula (I) may be dimerized.In certain embodiments, Xaa¹ is a 2-methyl benzoyl moiety.

In particular embodiments of the Formula (II) or Formula (VI) peptides(e.g. peptide monomers or peptide dimers or subunits thereof) of thepresent invention, Xaa¹ is a modified HomoSer or a modified Ser groupthat is capable of forming a thioether bond with Xaa⁷ and Xaa⁷ is Cys,Pen, D-Cys, Homo Cys. The N-terminal residue further comprises amodifying group or suitable linker moiety, e.g., a modifying group orlinker selected from the group consisting of DIG, PEG4, PEG13, PEG25,PEG1K, PEG2K, PEG4K, PEG5K, Polyethylene glycol having molecular weightfrom 400 Da to 40,000 Da, PEG having a molecular weight of 40,000 Da to80,000 Da, IDA, Ac-IDA, ADA, Glutaric acid, Succinic acid, Isophthalicacid, 1,3-phenylenediacetic acid, 1,4-phenylenediacetic acid,1,2-phenylenediacetic acid, AADA, suitable aliphatic acids, suitablearomatic acids, heteroaromatic acids. Examples of other linkers aredescribed herein and include but are not limited to those shown in Table3.

For each embodiment of Formula (II), Xaa² is selected from the groupconsisting of N(alpha)-Me-Arg, Arg, HArg, Dap, Dab, Arg-Me-sym,Arg-Me-asym, 4-Guan, Cit, Cav, and suitable isostere replacements. Insome embodiments, Xaa² is N(alpha)Methylated. Preferably, Xaa² isN-Me-Arg. In other embodiments, preferably Xaa² is Arg.

For each embodiment of Formula (II), Xaa³ is selected from the groupconsisting of Ser, Gly, and suitable isostere replacements. Preferably,Xaa³ is Ser.

For each embodiment of Formula (II), Xaa⁴ is selected from the groupconsisting of Asp, N-Me-Asp, Asp(OMe), D-Asp, and a suitable isosterereplacements. In some embodiments, Xaa⁴ is N(alpha)Methylated. In someembodiments, Xaa⁴ is Asp or N-Me-Asp. In some embodiments, Xaa⁴ is Asp.

For each embodiment of Formula (II), Xaa⁵ is selected from the groupconsisting of Thr, Gln, Ser, Asp, Pro, Gly, His, Ala, Ile, Phe, Lys,Arg, Asn, Glu, Val, Tyr, Trp, Leu, Met, and N-Methyl amino acidsincluding N-Me-Thr, and suitable isostere replacements. In someembodiments, Xaa⁵ is N(alpha)Methylated. In some embodiments, Xaa⁵ isselected from the group consisting of Thr, Gln, Ser, Asp, Gly, His, Ala,Ile, Phe, Lys, Arg, Asn, Glu, Val, Tyr, Trp, Leu, Met, and N-Methylamino acids including N-Me-Thr, and suitable isostere replacements.Preferably, Xaa⁵ is Thr.

For each embodiment of Formula (II), Xaa⁶ is selected from the groupconsisting of Gln, Asn, Asp, Pro, Gly, Ala, Phe, Leu, Glu, Ile, Val,HLeu, n-Butyl Ala, n-Pentyl Ala, n-Hexyl Ala, Nle, cyclobutyl-Ala,N-Me-Leu, and suitable isostere replacements. In some embodiments, Xaa⁶is selected from the group consisting of Gln, Asn, Asp, Pro, Gly, Ala,Phe, Leu, Glu, Ile, Val, HLeu, n-Butyl Ala, n-Pentyl Ala, n-Hexyl Ala,Nle, cyclobutyl-Ala, N-Me-Leu, and suitable isostere replacements. Insome embodiments, Xaa⁶ is N(alpha)Methylated. Preferably, Xaa⁶ is Leu.

For each embodiment of Formula (II), Xaa⁷ is selected from the groupconsisting of Cys, N-Me-Cys, D-Cys, HCys, Pen, and D-Pen. Preferably, inone embodiment Xaa⁷ is Pen. In another embodiment, Xaa⁷ is preferablyCys.

For each embodiment of Formula (II), Xaa⁸ is selected from the groupconsisting of Gly, Gln, Asn, Asp, Ala, Ile, Leu, Val, Met, Thr, Lys,Trp, Tyr, His, Glu, Ser, Arg, Pro, Phe, Sar, 1-Nal, 2-Nal, D-1-Nal,D-2-Nal, D-Phe, D-Tyr, HPhe, Phe(4-F), O-Me-Tyr, dihydro-Trp, Dap, Dab,Dab(Ac), Orn, D-Orn, N-Me-Orn, N-Me-Dap, D-N-Me-Lys, D-Dap, D-Dab, Bip,Ala(3,3diphenyl), Biphenyl-Ala, aromatic ring substituted Phe, aromaticring substituted Trp, aromatic ring substituted His, hetero aromaticamino acids, N-Me-Lys, N-Me-Lys(Ac), 4-Me-Phe, and corresponding D-aminoacids and suitable isostere replacements. In other embodiments, Xaa⁸ isN(alpha)Methylated. Further, in some embodiments Xaa⁸ is acylated. Insome embodiments of peptide monomers or peptide dimers described herein,Xaa⁸ is absent.

In particular embodiments of peptide dimer subunits of Formula (II) orFormula (VI), Xaa⁹⁻¹¹ are absent, and Xaa⁸ is the C-terminus of thesubunit. When Xaa⁸ is the C-terminus of the subunit, Xaa⁸ may bemodified to include a suitable linker moiety in accordance with thepresent invention.

In some embodiments of the peptide monomers and dimer subunits ofFormula (II) or Formula (VI), Xaa⁹ is absent, or Xaa⁹ is selected fromthe group consisting of Glu, Amide, Lys, Gln, Pro, Gly, His, Ala, Ile,Phe, Lys, COOH, Arg, Leu, Val, Tyr, Trp, Met, Gla, Ser, Asn, D-Glu,β-HGlu, 2-Nal, 1-Nal, D-1-Nal, D-2-Nal, D-Phe, D-Tyr, D-Asp, Bip,β-HPhe, β-Glu, D-Tyr, D-Lys, Dap, Dab, Orn, D-Orn, N-Me-Orn, N-Me-Dap,N-Me-Dab, N-Me Lys, D-N-Me-Lys D-Dap, D-Dab, suitable isosteres, andcorresponding D-amino acids. In particular embodiments of peptidemonomer or dimer subunits described herein, Xaa⁹ is absent or COOH. Incertain embodiments, Xaa⁹ is Glu, D-Glu, β-HGlu, or Asp.

In some embodiments of peptide dimer subunits, when Xaa¹⁰ and Xaa¹¹ areabsent, Xaa⁹ is the C-terminus of the subunit. When Xaa⁹ is theC-terminus of the subunit, Xaa⁹ may be modified to include a suitablelinker moiety in accordance with the present invention.

For each embodiment of Formula (II) or Formula (VI), Xaa¹⁰ may beabsent, or Xaa¹⁰ is selected from the group consisting of Gln, Pro, Gly,His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser, Asn,Gla, Dap, Dab, Orn, D-Orn, D-Lys, N-Me-Orn, N-Me-Dap, N-Me-Dab,D-N-Me-Lys N-Me-Lys, D-Dap, D-Dab, suitable isosteres, and correspondingD-amino acids. In at least one embodiment, Xaa¹⁰ is Lys. Further stillin some embodiments Xaa¹⁰ is D-Lys. In particular embodiments of peptidemonomers or peptide dimers described herein, Xaa¹⁰ is COOH or CONH₂.

In certain embodiments of peptide monomers or peptide dimer subunitscomprising Formula (II) or Formula (VI), when Xaa¹¹ is absent, Xaa¹⁰ isthe C-terminus. When Xaa¹⁰ is the C-terminus of the subunit, Xaa¹⁰ maybe modified to include a suitable linker moiety in accordance with thepresent invention. Further, in some embodiments, Xaa¹¹ is absent, orselected from the group consisting of Gln, Pro, Gly, His, Ala, Ile, Phe,Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser, Asn, Gla, Dap, Dab, Orn,D-Orn, D-Lys, N-Me-Orn, N-Me-Dap, N-Me-Dab, D-N-Me-Lys N-Me-Lys, D-Dap,D-Dab, suitable isosteres, and corresponding D-amino acids. In at leastone embodiment, Xaa¹⁰ is Lys. Further still in some embodiments Xaa¹⁰ isD-Lys. In some embodiments of peptide monomers, Xaa¹⁰ is COOH or CONH₂.

In certain embodiments of peptide monomers or peptide dimer subunits,Xaa¹¹ is the C-terminus. When Xaa¹¹ is the C-terminus of the subunit,Xaa¹¹ may be modified to include a linker moiety in accordance with thepresent invention.

In at least one embodiment of peptide monomers of the present invention,Xaa⁸⁻¹¹ are absent, whereby Xaa⁷ is the C-terminus.

In particular embodiments of peptide monomer and dimer subunitscomprising Formula (II), when Xaa⁹⁻¹¹ are absent, Xaa⁸ is theC-terminus. Similarly, in certain embodiments, when Xaa¹⁰ and Xaa¹¹ areabsent, Xaa⁹ is the C-terminus. Further, when Xaa¹¹ is absent, Xaa¹⁰ isthe C-terminus. In some embodiments, the C-terminus of the thioetherpeptide is modified to include a modifying group in accordance with thepresent invention. In some embodiments, the C-terminus of the thioetherpeptide monomer or dimer subunit comprises NH₂ or OH.

In particular embodiments of any of the peptides herein, including thosecomprising a structure of any one of Formulas (II), (II-A), (A), (III),or (IV) or Formula (VI), the thioether bond is in the reverse order,such that the amino acid residues and chemical moieties shown in Xaa¹are instead present in Xaa⁷, and the amino acid resides shown at Xaa⁷are instead present at Xaa¹. In this reverse orientation, the amino acidor chemical moiety at position Xaa⁷ is one that comprises a free amine.

In certain embodiments peptides comprising Formula (II) or Formula (VI):

Xaa¹ is a 2-Me-benzoyl group capable of forming a thioether bond withXaa⁷;

Xaa² is selected from the group consisting of N(alpha)-Me-Arg, Arg,HArg, Dap, Dab, Arg-Me-sym, Arg-Me-asym, 4-Guan, Cit, Cav, and suitableisostere replacements;

Xaa³ is selected from the group consisting of Ser, Gly, and suitableisostere replacements;

Xaa⁴ is selected from the group consisting of Asp, N-Me-Asp, Asp(OMe),D-Asp, and a suitable isostere replacements;

Xaa⁵ is selected from the group consisting of Thr, Gln, Ser, Asp, Pro,Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu, Val, Tyr, Trp, Leu, Met,and N-Methyl amino acids including N-Me-Thr, and suitable isosterereplacements;

Xaa⁶ is selected from the group consisting of Gln, Asn, Asp, Pro, Gly,Ala, Phe, Leu, Glu, Ile, Val, HLeu, n-Butyl Ala, n-Pentyl Ala, n-HexylAla, Nle, cyclobutyl-Ala, N-Me-Leu, and suitable isostere replacements;

Xaa⁷ is selected from the group consisting of Cys, N-Me-Cys, D-Cys,HCys, Pen, and D-Pen;

Xaa⁸ is selected from the group consisting of absent, Gly, Gln, Asn,Asp, Ala, Ile, Leu, Val, Met, Thr, Lys, Trp, Tyr, His, Glu, Ser, Arg,Pro, Phe, Sar, 1-Nal, 2-Nal, HPhe, Phe(4-F), O-Me-Tyr, dihydro-Trp, Dap,Dab, Dab(Ac), Orn, D-Orn, N-Me-Orn, N-Me-Dap, D-Dap, D-Dab, Bip,Ala(3,3diphenyl), Biphenyl-Ala, aromatic ring substituted Phe, aromaticring substituted Trp, aromatic ring substituted His, hetero aromaticamino acids, N-Me-Lys, N-Me-Lys(Ac), 4-Me-Phe, and corresponding D-aminoacids and suitable isostere replacements;

Xaa⁹ is selected from the group consisting of absent, Glu, Amide, Lys,COOH, CONH₂, Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr,Trp, Met, Gla, Ser, Asn, D-Glu, β-HGlu, 2-Nal, 1-Nal, D-Asp, Bip,β-HPhe, β-Glu, D-Tyr, D-Lys, Dap, Dab, Orn, D-Orn, N-Me-Orn, N-Me-Dap,N-Me-Dab, N-Me Lys, D-Dap, D-Dab, suitable isosteres, and correspondingD-amino acids;

Xaa¹⁰ is selected from the group consisting of absent, Gln, Pro, Gly,His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser, Asn,Gla, Dap, Dab, Orn, D-Orn, D-Lys, N-Me-Orn, N-Me-Dap, N-Me-Dab,N-Me-Lys, D-Dap, D-Dab, COOH, CONH₂, suitable isosteres, andcorresponding D-amino acids; and

Xaa¹¹ is selected from the group consisting of absent, Gln, Pro, Gly,His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser, Asn,Gla, Dap, Dab, Orn, D-Orn, D-Lys, N-Me-Orn, N-Me-Dap, N-Me-Dab,N-Me-Lys, D-Dap, D-Dab, COOH, CONH₂, suitable isosteres, andcorresponding D-amino acids, wherein the peptide further comprises athioether bond between Xaa¹ and Xaa⁷.

Another aspect of the present invention relates to a thioether peptidemonomer or each subunit of a dimer compound comprising the structureaccording to Formula (II-A) (SEQ ID NO: 45),

-   -   Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹        (Formula II-A)), or a pharmaceutically acceptable salt thereof,        wherein the peptide comprises a thioether bond between Xaa¹ and        Xaa⁷, wherein

Xaa¹ (or the N-terminus) of the peptide represented by Formula (II-A)comprises a group, e.g., optionally an aromatic group, that is capableof forming a thioether bond with Xaa⁷. In some embodiments, Xaa¹comprises a 2-methylbenzoyl moiety forming an amide bond with Xaa², andfurther comprising a methyl group forming a thioether bond with Xaa⁷.The 2-methylbenzoyl moiety further comprises substituent R-groupsrepresented by R1-R4; in some instances, at least one substituentR-group of Xaa¹ is a free amine, whereby the N-terminus of the thioetherpeptide of Formula (II-A) may be extended; in other instances, one ormore substituent groups represented by R1-R4 is selected from the groupconsisting of hydrogen, a methyl group, a fluorocarbon group, ahydrocarbon, Cl, CF3, OMe, OEt, CONH₂, an aromatic group, a smallpegylation group, a terminal modifying group, an acylation, a freeamine, and an acid. In particular embodiments, Formula (II-A) isdirected to a peptide monomer or peptide dimer subunit and Xaa¹ is amodified Ser or a modified Homo-Ser, e.g., Homo-Ser-Cl. In someembodiments, Formula (II-A) is directed to a peptide dimer subunit andXaa⁴ is modified Homo-Ser, and Xaa¹⁰ is Cys, D-Cys, or HomoCys.

For each embodiment of Formula (II-A), Xaa² is selected from the groupconsisting of N(alpha)-Me-Arg, Arg, HArg, Dap, Dab, Arg-Me-sym,Arg-Me-asym, 4-Guan, Cit, Cav, and suitable isostere replacements. Insome embodiments, Xaa² is N(alpha)Methylated. Preferably, Xaa² isN-Me-Arg. In other embodiments, preferably Xaa² is Arg.

For each embodiment of Formula (II-A), Xaa³ is selected from the groupconsisting of Ser, Gly, Thr, Ile and suitable isostere replacements.Preferably, Xaa³ is Ser.

For embodiments of Formula (II-A) directed to peptide monomers, Xaa⁴ isselected from the group consisting of Asp, N-Me-Asp, Asp(OMe), D-Asp,and a suitable isostere replacements. For embodiments of Formula (II-A),Xaa⁴ is selected from the group consisting of Asp, N-Me-Asp, D-Asp, anda suitable isostere replacements. In some embodiments of peptidemonomers and dimer subunits, Xaa⁴ is N(alpha)Methylated. Preferably,Xaa⁴ is Asp.

For each embodiment of Formula (II-A), Xaa⁵ is selected from the groupconsisting of Thr, Gln, Ser, Asp, Pro, Gly, His, Ala, Ile, Phe, Lys,Arg, Asn, Glu, Val, Tyr, Trp, Leu, Met, and N-Methyl amino acidsincluding N-Me-Thr, and suitable isostere replacements. In someembodiments, Xaa⁵ is N(alpha)Methylated. Preferably, Xaa⁵ is Thr.

For each embodiment of Formula (II-A), Xaa⁶ is selected from the groupconsisting of Gln, Asn, Asp, Pro, Gly, Ala, Phe, Leu, Glu, Ile, Val,HLeu, n-Butyl Ala, n-Pentyl Ala, n-Hexyl Ala, Nle, cyclobutyl-Ala,N-Me-Leu, and suitable isostere replacements. In some embodiments, Xaa⁶is N(alpha)Methylated. In some embodiments, Xaa⁶ is Leu.

For each embodiment of Formula (II-A), Xaa⁷ is selected from the groupconsisting of Cys, N-Me-Cys, D-Cys, HCys, Pen, D-Pen and Pen(=O).Preferably, in one embodiment Xaa⁷ is Pen. In another embodiment, Xaa⁷is preferably Cys. In particular embodiments of peptides (e.g. peptidemonomers, dimers or subunits thereof) of Formula (II-A), Xaa⁷ is capableof forming a thioether bond with Xaa¹. In some embodiments of peptides(e.g. peptide monomers, dimers or subunits thereof) of Formula (II-A),Xaa⁷ is Cys, D-Cys or HomoCys.

For each embodiment of Formula (II-A), Xaa⁸ is absent, or Xaa⁸ isselected from the group consisting of Gly, Gln, Asn, Asp, Ala, Ile, Leu,Val, Met, Thr, Lys, Trp, Tyr, His, Glu, Ser, Arg, Pro, Phe, Sar, 1-Nal,2-Nal, D-1-Nal, D-2-Nal, D-Phe, D-Tyr, HPhe, Phe(4-F), O-Me-Tyr,dihydro-Trp, Dap, Dab, Dab(Ac), Orn, D-Orn, N-Me-Orn, N-Me-Dap,D-N-Me-Lys, D-Dap, D-Dab, Bip, Ala(3,3diphenyl), Biphenyl-Ala,Phe(4tBu), Phe(4-OMe), Phe(4-COOH), Phe(2-carbomyl), Phe(3-carbomyl),Phe(CF3), Phe(2,4-diCl), Phe(3,4-diCl), Aic, N-Me-Tyr, N-Me-Phe, Tic,Phe(4CF3), aromatic ring substituted Phe, aromatic ring substituted Trp,aromatic ring substituted His, hetero aromatic amino acids, N-Me-Lys,N-Me-Lys(Ac), 4-Me-Phe, and corresponding D-amino acids and suitableisostere replacements. In other embodiments, Xaa⁸ is N(alpha)Methylated.Further, in some embodiments Xaa⁸ is acylated.

In some embodiments of Formula (II-A), Xaa⁹ is absent, or Xaa⁹ isselected from the group consisting of Glu, Amide, Lys, COOH, Gln, Pro,Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Gla, Ser,Asn, D-Glu, β-HGlu, 2-Nal, 1-Nal, D-1-Nal, D-2-Nal, D-Phe, D-Tyr, D-Asp,Bip, β-HPhe, β-Glu, D-Tyr, D-Lys, Dap, Dab, Orn, D-Orn, N-Me-Orn,N-Me-Dap, N-Me-Dab, N-Me Lys, D-N-Me-Lys D-Dap, D-Dab, O-Me-Glu,suitable isosteres, and corresponding D-amino acids. Preferably, Xaa⁹ isGlu, D-Glu, β-HGlu, Asp, D-His, F(4-COOH), Tic, D-Trp, D-Leu, D-Arg,D-Thr.

For particular embodiments of Formula (II-A), Xaa¹⁰ may be absent or anyamino acid. For certain embodiments, Xaa¹⁰ may be absent or Xaa¹⁰ isselected from the group consisting of Gln, Pro, Gly, His, Ala, Ile, Phe,Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser, Asn, Gla, Dap, Dab, Orn,D-Orn, D-Lys, N-Me-Orn, N-Me-Dap, N-Me-Dab, D-N-Me-Lys N-Me-Lys, D-Dap,D-Dab, COOH, CONH2, suitable isosteres, and corresponding D-amino acids.In at least one embodiment, Xaa¹⁰ is Lys. Further still in someembodiments Xaa¹⁰ is D-Lys.

Further, in particular embodiments of Formula (II-A) directed to peptidemonomers, Xaa¹¹ is absent or any amino acid. In certain embodimentsdirected to peptide monomers, Xaa¹¹ is selected from the groupconsisting of Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val,Tyr, Trp, Met, Glu, Ser, Asn, Gla, Dap, Dab, Orn, D-Orn, D-Lys,N-Me-Orn, N-Me-Dap, N-Me-Dab, D-N-Me-Lys, N-Me-Lys, D-Dap, D-Dab, COOH,CONH₂, suitable isosteres, and corresponding D-amino acids. In at leastone embodiment, Xaa¹¹ is Lys. Further still in some embodiments Xaa¹¹ isD-Lys.

In particular embodiments of Formula (II-A) directed to peptide dimersubunits, Xaa¹¹ is absent or selected from the group consisting of Gln,Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu,Ser, Asn, Gla, Dap, Dab, Orn, D-Orn, D-Lys, N-Me-Orn, N-Me-Dap,N-Me-Dab, D-N-Me-Lys, N-Me-Lys, D-Dap, D-Dab, Cys, HomoSys, Pen,suitable isosteres, and corresponding D-amino acids, and amino acidscomprising a free amine group. In at least one embodiment, Xaa¹¹ is Lys.Further still in some embodiments Xaa¹¹ is D-Lys. In at least oneembodiment, Xaa¹¹ is the C-terminus. When Xaa¹¹ is the C-terminus of thesubunit, Xaa¹¹ may be modified to include a linker moiety in accordancewith the present invention.

In particular embodiments of Formula (II-A), Xaa⁹ is not O-Me-Glu, andit absent or selected from the group consisting of Glu, Amide, Lys,COOH, Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp,Met, Gla, Ser, Asn, D-Glu, β-HGlu, 2-Nal, 1-Nal, D-1-Nal, D-2-Nal,D-Phe, D-Tyr, D-Asp, Bip, β-HPhe, β-Glu, D-Tyr, D-Lys, Dap, Dab, Orn,D-Orn, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me Lys, D-N-Me-Lys D-Dap, D-Dab,O-Me-Glu, suitable isosteres, and corresponding D-amino acids.

In particular embodiments of peptide monomers and dimer subunits, e.g. mthose of Formula (II) or (VI), Xaa⁸⁻¹¹ are absent, whereby Xaa⁷ is theC-terminus. When Xaa⁹⁻¹¹ are absent, Xaa⁸ is the C-terminus. Similarly,when Xaa¹⁰ and Xaa¹¹ are absent, Xaa⁹ is the C-terminus. Further, whenXaa¹¹ is absent, Xaa¹⁰ is the C-terminus. In certain embodiments,Xaa⁸⁻¹⁰ are absent, and Xaa¹¹ is the C-terminus. In certain embodiments,Xaa⁸ is present, Xaa⁹⁻¹⁰ are absent and Xaa¹¹ is the C-terminus. Incertain embodiments, Xaa⁸ and Xaa⁹ are present, Xaa¹⁰ is absent andXaa¹¹ is the C-terminus. In some embodiments of peptide monomers ordimers, the C-terminus of the thioether peptide is modified to include amodifying group or linker in accordance with the present invention.

For certain embodiments of Formula (II-A), a thioether bond existsbetween Xaa¹ and Xaa⁷. Thus, the thioether peptides of the presentinvention may be cyclized through a thioether bond. In one embodiment,Xaa⁷ is Cys. In another embodiment, preferably Xaa⁷ is Pen. In otherembodiments, Xaa⁷ is D-Cys or homo-Cys. In certain embodiments, Xaa¹ isHomo-Ser-Cl, and Xaa7 is Cys, D-Cys or HomoCys.

In some embodiments of peptide monomer, the C-terminal residue ofFormula (II) or (II-A) further comprises a modifying group selected fromthe group consisting of DIG, PEG4, PEG13, PEG25, PEG1K, PEG2K, PEG4K,PEG5K, Polyethylene glycol having molecular weight from 400 Da to 40,000Da, PEG having a molecular weight of 40,000 Da to 80,000 Da, IDA,Ac-IDA, ADA, Glutaric acid, Isophthalic acid, 1,3-phenylenediaceticacid, 1,4-phenylenediacetic acid, 1,2-phenylenediacetic acid, AADA,suitable aliphatic acids, suitable aromatic acids, heteroaromatic acids.In some embodiments, the C-terminus of the thioether peptide comprisesNH₂ or OH.

Some embodiments of the peptide monomers of the present inventioncomprise a peptide molecule comprising an N(alpha)-Me-Arg residue, asrepresented by at least one of SEQ ID NOs: 1-32.

In one embodiment, a thioether peptide of the present inventioncomprises one or two peptide dimer subunits or a peptide monomer ofFormula (A) (SEQ ID NO: 48):

Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰  (Formula (A)),

or a pharmaceutically acceptable salt thereof, wherein

Xaa¹ comprises an aromatic group capable of forming a thioether bondwith Xaa7, such as a 2-methylbenzoyl moiety;

Xaa² is N-methyl-Arg;

Xaa³ is Ser, Gly, Thr, or Ile; and

wherein in some embodiments if Formula (A) is directed to a peptidemonomer then Xaa³ is Ser, Gly, Thr, or Ile; and

wherein in other embodiments if Formula (A) is directed to a peptidedimer subunit then Xaa³ is Ser; and

Xaa⁴ is Asp;

Xaa⁵ is Thr;

Xaa⁶ is Leu or Nle;

Xaa⁷ is Cys, D-Cys, Hcys, or Pen;

Xaa⁸ is Trp, Tic, Bip, 1-Nal, 2-Nal, Phe(4tBu), or Phe(4-COOH);

Xaa⁹ is Glu, β-homo-Glu, or D-Glu;

Formula (A) is directed to a peptide monomer and Xaa¹⁰ is any aminoacid; or Formula (A) is directed to a peptide dimer subunit, and Xaa¹⁰is Lys, D-Lys, N-Me-Lys or D-N-Me-Lys; and

wherein the peptide molecule comprises a thioether bond between Xaa¹ andXaa⁷.

In particular embodiments of Formula (A), Xaa¹⁰ is D-Lys or N-Me-Lys.

In certain embodiments, Xaa¹⁰ or the C-terminus of the peptide comprisesan NH₂ or an OH.

In certain embodiments of peptide monomers, a free amine in theC-terminal amino acid is capped, e.g., with an acetyl group.

Illustrative thioether peptide dimers (and subunits thereof) and peptidemonomers of the present invention are shown in the accompanying figuresand sequence listing.

In certain embodiments, a thioether peptide monomer, dimer or peptidesubunit of a dimer, optionally a homodimer, of the present inventioncomprises Formula (III) (SEQ ID NO: 46):

Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa6-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰  (Formula (III)

or a pharmaceutically acceptable salt thereof, wherein the thioetherpeptide comprises a thioether bond between Xaa¹ and Xaa⁷ in the peptidemonomer or in one or both peptide monomer subunits, wherein the twosubunits of Formula (III) of a peptide dimer are dimerized at theirC-termini via a linker, e.g., DIG, and wherein

Xaa¹ is 2-methylbenzoyl;

Xaa² is N-Me-Arg;

Xaa³ is Ser, Gly, Thr, or Ile; or

Xaa⁴ is Asp;

Xaa⁵ is Thr; and

Xaa⁶ is Leu or Nle; or

Xaa⁷ is Pen, Cys or d-Cys; or

Xaa⁸ is Phe, D-Phe, Tyr, Bip, Tic, 1-Nal, 2-Nal, or Trp;

Xaa⁹ is D-Glu, Glu, Tyr, b-homo-Glu, or 2-Nal; and

Xaa¹⁰ is D-Lys, N-Me-D-Lys, Dap, Phe, D-Phe or absent.

In certain embodiments, Formula (III) is directed to a peptide monomerwherein:

Xaa¹ is 2-methylbenzoyl;

Xaa² is N-Me-Arg;

Xaa³ is Ser, Gly, Thr, or Ile;

Xaa⁴ is Asp;

Xaa⁵ is Thr;

Xaa⁶ is Leu or Nle;

Xaa⁷ is Pen, Cys or d-Cys;

Xaa⁸ is Phe, D-Phe, Tyr, 1-Nal, 2-Nal, or Trp;

Xaa⁹ is D-Glu, Glu, Tyr, b-homo-Glu, or 2-Nal; and

Xaa¹⁰ is D-Lys, N-Me-D-Lys, Dap, Phe, D-Phe or absent.

In certain embodiments, Formula (III) is directed to a peptide dimersubunit wherein:

Xaa¹ is 2-methylbenzoyl;

Xaa² is N-Me-Arg;

Xaa³ is Ser;

Xaa⁴ is Asp;

Xaa⁵ is Thr;

Xaa⁶ is Leu;

Xaa⁷ is Pen or, Cys;

Xaa⁸ is Phe, Tyr, Bip, Tic, 2-Nal, or Trp;

Xaa⁹ is D-Glu; and

Xaa¹⁰ is D-Lys.

In certain embodiments of peptide monomers, Xaa¹⁰ is acetylated orcomprises a modifying group, e.g., PEG8.

In certain embodiments, the C-terminus of a peptide monomer or subunitof a peptide dimer comprises an NH₂ or an OH. In particular embodiments,the C-terminus of a peptide dimer subunit comprises an NH₂ or an OHeither before or after dimerization.

In certain embodiments, a thioether peptide, e.g. a peptide monomer orpeptide dimer, optionally a homodimer, of the present inventioncomprises Formula (IV) (SEQ ID NO: 47):

Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa6-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰  (Formula (IV))

or a pharmaceutically acceptable salt thereof, wherein the thioetherpeptide comprises a thioether bond between Xaa¹ and Xaa⁷ in the peptidemonomer or in one or both peptide subunits of a peptide dimer, whereinthe two subunits of Formula (IV) are dimerized at their C-termini via alinker, e.g., DIG, and wherein

Xaa¹ is 2-methylbenzoyl;

Xaa² is N-Me-Arg;

Xaa³ is Ser;

Xaa⁴ is Asp;

Xaa⁵ is Thr;

Xaa⁶ is Leu or Nle;

Xaa⁷ is Pen, Cys, homoCys, Pen(=O), or D-Cys; wherein in certainembodiments, if Formula (IV) is directed to a peptide monomer, then Xaa⁷is Pen, Cys, homoCys, or D-Cys;

Xaa⁸ is Phe, D-Phe, Tyr, D-Tyr, His, Bip, Tic, 1-Nal, 2-Nal, F(CH3),F(2,4-diCl), F(3,4-diCl), Aic, N-Me-Tyr, N-Me-Phe, F(2-carbomyl),F(3-carbomyl), F(4-COOH), F(4OMe), F(4tBu), F-(4-F), F(4CF3), or Trp;and

Xaa⁹ is absent, Glu, β-homo-Glu, Bip, O-Me-Glu, D-Lys, D-Phe, Tyr,2-Nal, D-Tyr, Pro, Tic, D-Glu, D-Thr, D-Arg, D-Leu, D-Trp, F(4-COOH),D-His, Pro, D-Pro, or E(OMe); wherein in some embodiments, if Formula(IV) is directed to a peptide dimer subunit, then Xaa⁹ is Glu,β-homo-Glu, Bip, O-Me-Glu, D-Lys, D-Phe, Tyr, 2-Nal, D-Tyr, Pro, Tic,D-Glu, D-Thr, D-Arg, D-Leu, D-Trp, F(4-COOH), D-His, Pro, D-Pro, orE(OMe);

wherein in some embodiments, if Formula (IV) is directed to a peptidemonomer, then Xaa¹⁰ is absent or any amino acid residue; and

wherein in other embodiments, if Formula (IV) is directed to a peptidedimer subunit, then Xaa¹⁰ is D-Lys, N-Me-Lys, N-Me-D-Lys, Lys, Dap, Dab,D-Dab, D-Dap, Orn N-Me-Orn, D-Orn.

In certain embodiments of the peptide monomer or peptide dimer, Xaa¹⁰ orthe C-terminal amino acid does not comprise a free amine. In particularembodiments of the peptide monomer or peptide dimer, Xaa¹⁰ is D-Lys,N-Me-Lys, N-Me-D-Lys, Dap, Phe, Ser, Glu, or absent.

In certain embodiments of Formulas (II), (II-A), (A), (III), (IV), (VI)or Formula (VI), Xaa⁸ may also be Bpa, Phe(3-Me), Phe(2-Me), Phe(2-CF3),or β-Me-Phe.

In certain embodiments of Formulas (II), (II-A), (A), (III), (IV), (VI)or Formula (VI), Xaa⁹ may also be N-Me-Glu, N-Me-Asp, or alpha-H-Glu.

In certain embodiments of Formulas (II), (II-A), (A), (III), (IV), (VI)or Formula (VI), e.g., when the peptide is a dimer, Xaa¹⁰ is selectedfrom the group consisting of: Lys, D-Lys, N-Me-Lys, D-N-Me-Lys, Orn,Dab, Dap, Homo-Lys, D-Dap, D-Dab, Cys, HomoCys, Pen, or D-Orn, while inother embodiments, Xaa¹⁰ is selected from D-Lys, N-Me-Lys, andD-N-Me-Lys.

In certain embodiments of the peptide monomers or peptide dimersdescribed herein, the N-terminus of the peptide is acylated.

In certain embodiments of the peptide monomers and dimer subunits, Xaa¹⁰or the C-terminus of each peptide or peptide subunit comprises an NH₂ oran OH. In certain embodiments of the peptide dimer subunits, theC-terminus of comprises an NH₂ or an OH either before or afterdimerization.

In certain embodiments of peptide monomers described herein, a freeamine in the C-terminal amino acid is capped, e.g., with an acetylgroup.

Particular aspects of the present invention relate to peptide inhibitorsof α4β7 comprising the following core consensus sequence (shown left toright from N-term to C-term):

(SEQ ID NO: 390) Y-(N-Me-Arg)-Ser-Glu-Thr-Leu-X

wherein Y is a 2-methyl benzoyl moiety capable of forming a thioetherbond with X, and wherein X is an amino acid residue selected from Pen,Cys, D-Cys and HomoCys. In particular embodiments, X is Pen. Inparticular embodiments, the core sequence comprises an intramolecularthioether bond between X and Y. In particular embodiments, the peptideinhibitor is a monomer. In particular embodiments, the peptide inhibitoris a dimer comprising two peptide monomer subunits, each comprising thiscore sequence. In particular embodiments, the monomer peptide inhibitorcomprises 7-15 amino acid residues. In particular embodiments, eachmonomer subunit of the dimer peptide inhibitor comprises 7-15 amino acidresidues. In certain embodiments, the two monomer subunits are linkervia their respective N- or C-termini. In particular embodiments, theyare linker by each of their C-termini. In certain embodiments, thepeptide inhibitor further comprises an aromatic amino acid immediatelydownstream of X. In particular embodiments, any of the peptidesdescribed herein may comprise this core sequence.

In some embodiments, the N- or C-terminal residue of Formula (I),Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI)Formula (I-A), Formula (II-A), Formula (A), or any of the other peptidemonomers or peptide subunits of dimer molecules described herein,further comprises a modifying group or suitable linker moiety selectedfrom the group consisting of DIG, PEG4, PEG13, PEG25, PEG1K, PEG2K,PEG4K, PEG5K, Polyethylene glycol having molecular weight from 400 Da to40,000 Da, PEG having a molecular weight of 40,000 Da to 80,000 Da, IDA,Ac-IDA, ADA, Glutaric acid, Isophthalic acid, 1,3-phenylenediaceticacid, 1,4-phenylenediacetic acid, 1,2-phenylenediacetic acid, AADA,suitable aliphatic acids, suitable aromatic acids, heteroaromatic acids.

Particular embodiments of the present invention relate to a peptidedimer comprising a linker. When the linker is IDA, ADA or any linkerwith free amine, it can be acylated with acylating organic compoundselected from the group consisting of 2-me-Trifluorobutyl,Trifluoropentyl, Acetyl, Octonyl, Butyl, Pentyl, Hexyl, Palmityl,Lauryl, Oleoyl, Lauryl, Trifluoromethyl butyric, cyclopentanecarboxylic, cyclopropylacetic, 4-fluorobenzoic, 4-fluorophenyl acetic,3-Phenylpropionic, tetrahedro-2H-pyran-4carboxylic, succinic acid, andglutaric acid, straight chain aliphatic acids with 10 to 20 carbonunits, cholic acid and other bile acids. In some instances small PEG(PEG4-PEG13), Glu, Asp, is used as spacer before acylations.

Some embodiments of the present invention comprise a peptide monomer ordimer molecule comprising an N(alpha)-Me-Arg residue, as represented byat least one of SEQ ID NOs: 1-23.

In certain embodiments, a peptide monomer or at least one subunit of apeptide dimer molecule of the present invention comprises, consistsessentially of, or consists of an amino acid sequence or structuredescribed herein, including any of the amino acid sequences shown in theaccompanying sequence listing or figures, with or without any indicatedN- or C-terminal modifications, linkers or modifying group. In certainembodiments, a peptide dimer molecule of the present invention comprisestwo peptide monomer subunits, each having an amino acid sequence orstructure described herein, including any of the amino acid sequencesshown in the accompanying sequence listing or figures, with or withoutany indicated N- or C-terminal modifications, linkers or modifyinggroup. In particular embodiments, a peptide monomer or one or both ofthe peptide monomer subunits present in a peptide dimer moleculeincludes a thioether intramolecular linkage, e.g., a thioether bondbetween two amino acids within the peptide or subunit. In particularembodiments, the peptide subunits of a peptide dimer molecule aredimerized via their N- or C-termini, e.g., using a suitable linker suchas DIG.

In certain embodiments of the peptide dimer molecules, the presentinvention includes a peptide subunit comprising, consisting essentiallyof, or consisting of an amino acid sequence or structure describedherein, including any of the amino acid sequences shown in theaccompanying sequence listing or figures, with or without any indicatedN- or C-terminal modifications, linkers or modifying group. In certainembodiments, the peptide subunit includes a thioether intramolecularlinkage, e.g., a thioether bond between two amino acids within thepeptide subunit. In particular embodiments, the peptide monomer subunitcomprises a linker moiety, e.g., DIG, at it N- or C-termini.

In certain embodiments of any of the peptide monomers or dimer peptidesubunits described herein, including those of Formula (I)-(VI) andTables 4 and 5, or of the figures herein, the peptide monomer or subunitcomprises a thioether bond. In certain embodiments, with respect toFormula (I) or (V), the thioether bond exists between Xaa⁴ and Xaa¹⁰,wherein with respect to Formulas (II)-(IV) and (VI), the thioether bondexists between Xaa¹ and Xaa⁷. In certain embodiments, the thioether isformed between a 2-methyl benzoyl moiety (e.g., at Xaa⁴ in Formula (I)or Xaa¹ in Formula (II)) and either Pen or Cys (e.g., at Xaa¹⁰ inFormula (I) or Xaa⁷ in Formula (II)). In particular embodiments, the2-methyl benzoyl moiety forms an amide bond with an adjacent amino acidresidue and comprises a methyl group forming a thioether bond with thePen or Cys residue.

In particular embodiments of any of the various Formulas describedherein, peptides having the same structure or sequence as disclosed inany one or more of PCT/US2013/064439, PCT/US2014/032391 orPCT/US2014/032392 are excluded. In other embodiments of the presentinvention, the peptides comprise a sequence or structure set forth inany of PCT/US2013/064439, PCT/US2014/032391 or PCT/US2014/032392.

Peptide Molecule Structure and Biological Activity

The present invention provides various novel antagonist peptide monomersand peptide dimers, including peptide monomers and dimer moleculesubunits which are cyclized through a thioether bond. These peptidemolecules have been tested to more clearly characterize the increasedaffinity for α4β7 binding, increased selectivity against α4β1, andincreased stability in simulated intestinal fluid (SIF) as well as ingastric environment under reduced conditions. These novel antagonistmolecules demonstrate high binding affinity with α4β7, therebypreventing binding between α4β7 and the MAdCAM ligand. Accordingly,these peptide molecules have shown to be effective in eliminating and/orreducing the inflammation process in various experiments.

The present invention thus provides various thioether peptide monomerand dimer molecules which bind or associate with the α4β7 integrin,e.g., in serum, SIF, or SGF, to disrupt or block binding between α4β7and the MAdCAM ligand. Some peptide monomer or peptide subunits of theinvention may be constructed solely of natural amino acids.Alternatively, the peptide monomer and dimer molecules may includenon-natural amino acids including, but not limited to, modified aminoacids and suitable aromatic acid groups, namely a 2-methylbenzoylmoiety. Modified amino acids include natural amino acids which have beenchemically modified to include a group, groups, or chemical moiety notnaturally present on the amino acid. The thioether peptide monomer anddimer molecules of the present invention may additionally includeD-amino acids.

In certain embodiments, peptide dimer and monomer molecules of thepresent invention inhibit or reduce binding between α4β7 and the MAdCAMligand. In certain embodiments, a peptide of the present inventionreduces binding of α4β7 and the MAdCAM ligand by at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, or at least 90% as compared to a negative control peptide. Methodsof determining binding are known in the art and described herein, andinclude ELISA assays, for example.

In certain embodiments, a peptide monomer or dimer molecule of thepresent invention has an IC50 of <500 nM, <250 nM, <100 nM, <50 nM, <25nM, or <10 nM. Methods of determining activity are known in the art andinclude any of those described in the accompanying Examples.

Some antagonist thioether cyclized peptide monomer and dimer moleculeshave been shown to be gastrointestinal stable and provide high levels ofspecificity and affinity for the α4β7 integrin. Some implementations ofthe present invention provide a peptide monomer or dimer moleculecomprising a half-life of greater than 180 minutes when exposed tosimulated intestinal fluids (SIF). Some implementations further providea thioether peptide monomer or dimer molecule comprising a half-lifefrom approximately 1 minute to approximately 180 minutes. Similarlythese peptides are stable to gastric environment under reducedconditions with half-life >120 min when tested in DTT (Dithiothreitol)assay.

In certain embodiments, a peptide monomer or dimer molecule of thepresent invention has increased stability, increased gastrointestinalstability, or increased stability in stimulated intestinal fluid (SIF),as compared to a control peptide. In particular embodiments, a controlpeptide is a peptide having the identical or a highly related amino acidsequence (e.g., >90% sequence identity) as the peptide monomer or dimermolecule, but which does not form a cyclized structure through athioether bond. In some embodiments relating to dimer molecules, thecontrol peptide is not dimerized. In particular embodiments, the onlydifference between the peptide monomer or dimer molecule and the controlpeptide is that the peptide comprises one or more amino acidsubstitutions that introduce one or more amino acid residues into thepeptide, wherein the introduced residue(s) forms a thioether bond withanother residue in the peptide.

Methods of determining the stability of a peptide are known in the art.In certain embodiments, the stability of a peptide (e.g. a peptidemonomer or dimer as described herein) is determined using an SIF assay,e.g., as described in the accompanying Examples. In particularembodiments, a peptide monomer or dimer molecule of the presentinvention has a half-life under a given set of conditions (e.g.,temperature) of greater than 1 minute, greater than 10 minutes, greaterthan 20 minutes, greater than 30 minutes, greater than 60 minutes,greater than 90 minutes, greater than 120 minutes, greater than 3 hours,or greater than four hours when exposed to SIF. In certain embodiments,the temperature is about 25° C., about 4° C., or about 37° C., and thepH is a physiological pH, or a pH about 7.4.

In some embodiments, the half-life is measured in vitro using anysuitable method known in the art, e.g., in some embodiments, thestability of a peptide monomer or dimer molecule of the presentinvention is determined by incubating the peptide with pre-warmed humanserum (Sigma) at 37° C. Samples are taken at various time points,typically up to 24 hours, and the stability of the sample is analyzed byseparating the peptide monomer or dimer from the serum proteins and thenanalyzing for the presence of the peptide monomer or dimer of interestusing LC-MS.

In some embodiments, a peptide monomer or dimer molecule of the presentinvention exhibits improved solubility or improved aggregationcharacteristics as compared to a control peptide. Solubility may bedetermined via any suitable method known in the art. In someembodiments, suitable methods known in the art for determiningsolubility include incubating peptides in various buffers (AcetatepH4.0, Acetate pH5.0, Phos/Citrate pH5.0, Phos Citrate pH6.0, Phos pH6.0, Phos pH 7.0, Phos pH7.5, Strong PBS pH 7.5, Tris pH7.5, Tris pH8.0, Glycine pH 9.0, Water, Acetic acid (pH 5.0 and other known in theart) and testing for aggregation or solubility using standardtechniques. These include, but are not limited to, visual precipitation,dynamic light scattering, Circular Dichroism and fluorescent dyes tomeasure surface hydrophobicity, and detect aggregation or fibrillation,for example. In some embodiments, improved solubility means the peptidemonomer or dimer is more soluble in a given liquid than is a controlpeptide.

In some embodiments, the peptide monomer and dimer molecules of thepresent invention have less degradation (i.e., more degradationstability), e.g., greater than or about 10% less, greater than or about20% less, greater than or about 30% less, greater than or about 40 less,or greater than or about 50% less degradation than a control peptide. Insome embodiments, degradation stability is determined via any suitablemethod known in the art. In some embodiments, suitable methods known inthe art for determining degradation stability include the methoddescribed in Hawe et al J Pharm Sci, VOL. 101, NO. 3, 2012, p 895-913,incorporated herein in its entirety. Such methods are in someembodiments used to select potent peptide monomer or dimer moleculeswith enhanced shelf lifes.

In some embodiments, peptide dimer or monomer molecules of the presentinvention have increased redox stability as compared to a controlpeptide. Methods of determining redox stability are described herein.

In certain embodiments, peptide dimer or monomer molecules of thepresent invention inhibit or reduce α4β7-mediated inflammation. Inrelated embodiments, peptide monomers or dimers of the present inventioninhibit or reduce α4β7-mediated secretion of one or more cytokines.Methods of determining inhibition of cytokine secretion and inhibitionof signaling molecules are known in the art.

In certain embodiments, peptide monomer or dimer molecules of thepresent invention demonstrate increased binding selectivity. In certaininstances, peptide monomers or dimers of the present invention binds toα4β7 with at least a two-fold, three-fold, five-fold, or ten-foldgreater affinity than the monomers or dimers bind to α4β1.

The peptide monomer or dimer molecules of the present inventiondemonstrate increased potency as a result of substituting variousnatural amino acyl residues with N-methylated analog residues. Inparticular embodiments, potency is measured as IC50 of binding to α4β7,e.g., determined as described herein, while in some embodiments, potencyindicates functional activity, e.g., according to a cell adhesion assayas described herein or a PBMC assay described herein. For example, SEQID NOs.: 1-32 represent peptide monomer or subunit sequences that aresubstituted with N(alpha)methylated arginine.

In particular embodiments, any of these superior characteristics of thepeptides of the present invention are measured as compared to a controlpeptide, e.g., a peptide shown in Table 8.

Referring now to FIG. 6 and Tables 5 and 7, charts are provided whichinclude various data illustrating increased potency and/or stability forvarious non-limiting sample thioether peptide dimer molecules inaccordance with the instant invention. Simulated Intestinal Fluid (SIF)Stability assays were performed for the majority of the dimer molecules.A selective sampling of these results is provided in FIG. 6. Indicatedthioether peptides in FIG. 6 represent a non-limiting, representativegroup of dimer peptides with stability of greater than 180 minutes(half-life) in SIF. These thioether dimer compounds further representIC50 values of less than 25 nM in ELISA as well as cell adhesion assays,further demonstrating their high selectivity for α4β7. For otherpeptides in FIG. 6, it is expected that they will have an IC50<50 nM inα4β7 ELISA or cell adhesion assays.

Referring now to FIGS. 7 and 8 and Tables 4 and 6, charts are providedwhich includes various data illustrating increased potency for variousnon-limiting illustrative thioether peptide monomers in accordance withthe instant invention. Potency assays were performed for all peptidemolecules represented by SEQ ID NOs: 22 and 23 and additional peptidesas shown. Selectivity assays (for α4β1) were performed for certainthioether peptides. A selective sampling of these results is provided inFIGS. 7 and 8. Improvements in potency for α4β7 were tested in bothELISA and cell adhesion assays.

According to the protocols discussed herein, applicant successfullysynthesized and purified all of the integrin antagonist thioetherpeptides (e.g. peptide monomers and peptide dimers) represented by SEQID NOs: 22 to 24 and additional peptides shown in Tables 4-7 and FIGS.6-8. The majority of these molecules were subjected to an α4β7-MAdCAMCompetition ELISA assay, an α4β1-VCAM Competition ELISA assay, and anα4β7-MadCAM cell adhesion assay. Results are provided in Tables 6-7 andFIGS. 6-8. The thioether peptides shown in FIG. 7 represent anon-limiting, representative group of peptides with IC50 values lessthan 50 nM in ELISA assays. The peptides further represent IC50 valuesof less than 300 nM in cell adhesion assays. For other peptides withdata not shown, it is expected that they will have an IC50<50 nM in α4β7ELISA or cell adhesion assays.

When Arg is replaced with N-Me-Arg, a significant improvement in potencyfor α4β7 was shown in both ELISA and cell adhesion assays.N(alpha)methylation further demonstrated increased molecular stability.One having skill in the art will appreciate that methylated isosteres ofarginine may further demonstrate similar increases in potency and/orstability.

Referring now to FIGS. 6 and 8 charts are provided which include dataillustrating increased stability for various non-limiting samplethioether peptide molecules in accordance with the instant invention.Simulated Intestinal Fluid (SIF) Stability assays were performed for themajority of the peptide molecules. A selective sampling of these resultsis provided in FIGS. 6 and 8. The thioether peptides in FIGS. 6 and 8represent a non-limiting, representative group of peptides withstability of greater than 180 minutes (half-life) in SIF.

Methods of Manufacture and Enhancing Peptide Stability

The peptides (e.g. peptide monomers or peptide dimers) of the presentinvention may be synthesized by techniques that are known to thoseskilled in the art. Such techniques include the use of commerciallyavailable robotic protein synthesizers (e.g. Symphony multiplex peptidesynthesizer from Protein Technologies). In some embodiments, novelpeptide monomers or dimer subunits are synthesized and purified usingtechniques described herein.

Certain aspects of the present invention contemplate peptides comprisingthioether bonds. Thioether bonds are cyclized covalent bonds formedbetween an upstream amino acid or aromatic acid group and a downstreamsulfur-containing amino acid or isotere thereof. Thioether bonds of thepresent invention may be generated using standard techniques in the art,including those described herein. Particular aspects contemplate thatthe generation of a thioether bond increases gastrointestinal stabilityof a peptide molecule. Thus, in particular embodiments, gastrointestinalstability of a peptide can be increased by cyclizing the peptide via athioether bond.

In some embodiments, monomeric subunits of the present invention may bedimerized to form homomeric or heteromeric dimer peptides through knowntechniques in the art. In certain embodiments, peptide subunitsdescribed herein are joined by linker moieties (e.g. linkers shown inTable 3) conjugated at the N or C-termini. A linker may be conjugated topeptide subunit at a C- or N-terminal free amine through techniquesknown in the art, including but not limited to techniques describedherein. Some embodiments contemplate that dimerization of the peptidemolecule increases stability, potency, and/or specificity as compared tonon-dimerized monomeric subunits of the peptide.

Certain aspects of the present invention contemplate amino acidsubstitutions that increase stability of a peptide monomer or peptidedimer in different contexts. Accordingly, in certain embodiments, thepresent invention includes modifying a peptide molecule, e.g., a peptidemolecule described herein or Substitutions may be performed by standardtechniques known to those of skill in the art. In some embodiments,stability of a peptide (e.g. a peptide monomer or dimer described hereinor in Dubree, et al., Selective α4β7 Integrin Antagonist and TheirPotential as Anti-inflammatory Agents, J. Med. Chem. 2002, 45,3451-3457) in simulated intestinal fluids (SIF) is increased bysubstituting N-Me-Arg for one or more unmethylated arginine residues. Inparticular embodiments, SIF or gastrointestinal stability of a peptideis increased by substituting Pen for one or more cysteine residues.Certain aspects of the present invention contemplate amino acidsubstitutions that increase redox stability (i.e. increasing theresistance of a peptide to a change in its oxidation state) of a peptidemonomer or peptide dimer described herein. In particular embodiments,redox stability is determined by an assay described herein. Inparticular embodiments, redox stability is increased by at least 20%, atleast 50%, at least 2-fold, at least 3-fold, at least 4-fold, at least5-fold, or at least 10-fold as compared to a control peptide.Substitutions may be performed by standard techniques known to those ofskill in the art. In some embodiments, redox or gastrointestinalstability of a peptide (e.g. peptide monomer or dimer described herein)is increased by substituting N-Me-Arg for one or more unmethylatedarginine residues.

In particular embodiments, the invention provides a method forstabilizing a peptide molecule, e.g., a peptide molecule describedherein, comprising cyclizing the peptide molecule by forming a thioetherbond between Xaa⁴ and Xaa¹⁰.

In certain embodiments, the invention includes a method for stabilizinga peptide molecule, e.g., of Formula (II), comprising: substituting Xaa¹with an aromatic acid group capable of forming a thioether bond withXaa⁷; substituting Xaa⁷ with an amino acid residue that is capable offorming a thioether bond with Xaa¹; and forming a thioether bond betweenXaa¹ and Xaa⁷ to provide a cyclized peptide. In certain embodiments,Xaa⁷ is selected from the group consisting of Cys, N-Me-Cys, D-Cys,HCys, Pen, and D-Pen. In certain embodiments, Xaa¹ is a 2-methylbenzoylmoiety. The same method applies to peptide molecules, e.g., of Formula(I), where Xaa4 and Xaa10 are substituted and cyclized instead of Xaa1and Xaa7, respectively.

Methods of Treatment and Pharmaceutical Compositions

In some embodiments, the present invention provides a method fortreating an individual or subject afflicted with a condition orindication characterized by integrin binding, wherein the methodcomprises providing or administering to the individual or subject anintegrin antagonist thioether peptide molecule described herein, e.g.,as represented by SEQ ID NOs: 1-384 or shown in Tables 5-7. Inparticular embodiments, the individual or subject is provided with oradministered with a pharmaceutical composition comprising the peptidemonomer or peptide dimer of the invention. In particular embodiments,subjects or individuals are mammals, e.g., humans or non-human mammals,such as a dog, cat or horse.

In one embodiment, a method is provided for treating an individual orsubject afflicted with a condition or indication characterized byinappropriate trafficking of cells expressing α4β7 to tissues comprisingcells expressing MAdCAM, comprising administering to the individual orsubject an α4β7-antagonist peptide molecule described herein, e.g., SEQID NOs: 1-384 or Tables 4 and 5, in an amount sufficient to inhibit(partially or fully) the trafficking of cells expressing α4β7 to tissuescomprising cells expressing MAdCAM.

In a further related embodiments, the present invention includes amethod for treating a condition in a subject or individual in needthereof, wherein the condition is treatable by reducing the activity(partially or fully) of α4β7 in the subject, comprising providing oradministering an α4β7-antagonist peptide molecule described herein tothe subject. In particular embodiments, the condition is an inflammatorycondition of the gastrointestinal system.

In a further related embodiments, the present invention includes amethod for treating a subject, e.g., a mammal or human, afflicted with acondition that is associated with a biological function α4β7, comprisingproviding or administering to the subject a thioether peptide moleculedescribed herein, e.g., a peptide monomer or peptide dimer having astructure of Formula (I) or (II), in an amount sufficient to inhibit(partially or fully) the biological function of α4β7 to tissuesexpressing MAdCAM. In particular embodiments, the subject is providedwith an effective amount of the peptide monomer or peptide dimersufficient to at least partially inhibit the biological function of α4β7to tissues expressing MAdCAM. In certain embodiments, the condition isinflammatory bowel disease.

In additional embodiments, the invention includes a method of treatingor preventing a disease or condition in a subject in need thereof,comprising providing or administering to the subject, e.g., a mammal, aneffective amount of a peptide dimer or peptide monomer described herein,wherein the disease or condition is selected from the group consistingof Inflammatory Bowel Disease (IBD) (including adult IBD, pediatric IBDand adolescent IBD), ulcerative colitis, Crohn's disease, Celiac disease(nontropical Sprue), enteropathy associated with seronegativearthropathies, microscopic colitis, collagenous colitis, eosinophilicgastroenteritis, radiotherapy, chemotherapy, pouchitis resulting afterproctocolectomy and ileoanal anastomosis, gastrointestinal cancer,pancreatitis, insulin-dependent diabetes mellitus, mastitis,cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronicsinusitis, asthma, primary sclerosing cholangitis, humanimmunodeficiency virus (HIV) infection in the GI tract, eosinophilicasthma, eosinophilic esophagitis, gastritis, colitis, microscopiccolitis and graft versus host disease (GVDH) (including intestinalGVDH). In particular embodiments of any of the methods of treatmentdescribed herein, the subject has been diagnosed with or is consideredto be at risk of developing one of these diseases or conditions.

In particular embodiments of any of the methods of treatment describedherein, the peptide molecule (or pharmaceutical composition comprisingthe peptide molecule) is administered to the individual by a form ofadministration selected from the group consisting of oral, intravenous,peritoneal, intradermal, subcutaneous, intramuscular, intrathecal,inhalation, vaporization, nebulization, sublingual, buccal, parenteral,rectal, vaginal, and topical.

In certain embodiments, the α4β7 integrin antagonist peptide moleculecomprises an increased half-life as compared to other peptides. Inparticular embodiments, the increased half-life is at least one day invitro or in vivo. In certain embodiments wherein the increased half-lifeis equal to or greater than a period consistent with no more frequentthan twice daily dosing in vivo, the α4β7 integrin antagonist peptidemolecule is provided in a pharmaceutical preparation that isadministered orally. In certain embodiments wherein the increasedhalf-life is from approximately 12 hours to greater than 24 in vivo, theα4β7 integrin antagonist peptide molecule is provided in apharmaceutical preparation that is administered parenterally. In certainembodiments when the increased half-life is from approximately 12 hoursto greater than 24 hours in vivo, the α4β7 integrin antagonist peptidemolecule is provided in a pharmaceutical preparation that isadministered topically.

In some embodiments, the present invention provides a method whereby apharmaceutical composition comprising an integrin antagonist thioetherpeptide molecule described herein, e.g., SEQ ID NOs: 1-384 or Tables 4or 5, is administered to a subject or patient as a first treatment. Inanother embodiment, the method further comprises administering to thesubject a second treatment, i.e., a second active agent. In anotherembodiment, the second treatment or active agent is administered to thesubject before and/or simultaneously with and/or after thepharmaceutical composition is administered to the subject. In otherembodiment, the second treatment or active agent comprises ananti-inflammatory agent. In another embodiment, the second treatment oractive agent (which may be present in a pharmaceutical composition)comprises an agent selected from the group consisting of non-steroidalanti-inflammatory drugs, steroids, and immune modulating agents. Inanother embodiment, the method comprises administering to the subject athird treatment.

The thioether peptide monomer and dimer molecules of the invention,including but not limited to those specified in the examples, possessintegrin-antagonist activity. In certain embodiments, peptide integrininhibitors (e.g. thioether peptide monomers and dimers described herein)are administered to a subject in need of treatment for InflammatoryBowel Disease (IBD), ulcerative colitis, Crohn's disease, Celiac disease(nontropical Sprue), enteropathy associated with seronegativearthropathies, microscopic or collagenous colitis, eosinophilicgastroenteritis, radio- and chemotherapy, or pouchitis resulting afterproctocolectomy and ileoanal anastomosis and various forms ofgastrointestinal cancer, osteoporosis, arthritis, multiple sclerosis,chronic pain, weight gain, and/or depression.

In another embodiment, peptide integrin inhibitors of the presentinvention are administered to a subject in need of treatment forpancreatitis, insulin-dependent diabetes mellitus, mastitis,cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronicsinusitis, asthma and/or graft versus host disease. In addition, thesepeptide monomer and dimer molecules may be useful in the prevention orreversal of these diseases when used in combination with currentlyavailable therapies, medical procedures, and therapeutic agents.

In one embodiment, a method is provided for treating an individual orsubject afflicted with a condition or indication characterized by α4β7integrin binding, wherein the method comprises administering to theindividual or subject an effective amount of an α4β7 integrin antagonistpeptide molecule described herein, e.g., SEQ ID NOs: 1-384 or Tables 4or 5. In some instances, an α4β7 integrin antagonist peptide moleculedescribed herein, e.g., corresponding to SEQ ID NOs: 1-384 or Tables 4or 5, and having high specificity for α4β7 is administered to anindividual as part of a therapeutic treatment for a condition orindication characterized by α4β7 integrin binding.

In particular embodiments, the peptide molecules of the presentinvention are present in a pharmaceutical composition further comprisingone or more pharmaceutically acceptable diluents, carriers, orexcipients. In particular embodiments, they are formulated as a liquidor solid. In particular embodiments, they are formulated as a tablet orcapsule, or as a liquid suspension. Some embodiments of the presentinvention further provide a method for treating an individual with anα4β7 integrin antagonist peptide molecule of the present invention thatis suspended in a sustained-release matrix. A sustained-release matrix,as used herein, is a matrix made of materials, usually polymers, whichare degradable by enzymatic or acid-base hydrolysis or by dissolution.Once inserted into the body, the matrix is acted upon by enzymes andbody fluids. A sustained-release matrix desirably is chosen frombiocompatible materials such as liposomes, polylactides (polylacticacid), polyglycolide (polymer of glycolic acid), polylactideco-glycolide (copolymers of lactic acid and glycolic acid)polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such as phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. On particularbiodegradable matrix is a matrix of one of either polylactide,polyglycolide, or polylactide co-glycolide (co-polymers of lactic acidand glycolic acid).

In some aspects, the invention provides a pharmaceutical composition fororal delivery. The various embodiments and thioether peptide moleculecompositions of the instant invention may be prepared for oraladministration according to any of the methods, techniques, and/ordelivery vehicles described herein. Further, one having skill in the artwill appreciate that the peptide molecule compositions of the instantinvention may be modified or integrated into a system or deliveryvehicle that is not disclosed herein, yet is well known in the art andcompatible for use in oral delivery of small peptide molecules.

Oral dosage forms or unit doses compatible for use with the peptides ofthe present invention may include a mixture of peptide active drugcomponents, and nondrug components or excipients, as well as othernon-reusable materials that may be considered either as an ingredient orpackaging. Oral compositions may include at least one of a liquid, asolid, and a semi-solid dosage forms. In some embodiments, an oraldosage form is provided comprising an effective amount of a thioetherpeptide molecule described herein, e.g., corresponding to any of SEQ IDNOs: 1-384 or Tables 4 or 5, wherein the dosage form comprises at leastone of a pill, a tablet, a capsule, a gel, a paste, a drink, and asyrup. In some instances, an oral dosage form is provided that isdesigned and configured to achieve delayed release of the thioetherpeptide molecule in the small intestine of the subject.

In one embodiment, an oral pharmaceutical composition comprising athioether peptide of the present invention comprises an enteric coatingthat is designed to delay release of the peptide molecule in the smallintestine. In at least some embodiments, a pharmaceutical composition isprovided which comprises a peptide molecule described herein, e.g.,corresponding to any of SEQ ID NOs: 1-384, or Tables 4 or 5, and aprotease inhibitor, such as aprotinin, in a delayed releasepharmaceutical formulation. In some instances it is preferred that apharmaceutical composition of the instant invention comprise an entericcoat that is soluble in gastric juice at a pH of about 5.0 or higher. Inat least one embodiment, a pharmaceutical composition is providedcomprising an enteric coating comprising a polymer having dissociablecarboxylic groups, such as derivatives of cellulose, includinghydroxypropylmethyl cellulose phthalate, cellulose acetate phthalate andcellulose acetate trimellitate and similar derivatives of cellulose andother carbohydrate polymers.

In one embodiment, a pharmaceutical composition comprising a thioetherpeptide molecule described herein, e.g., corresponding to any of SEQ IDNOs: 1-384 or Tables 4 and 5, is provided in an enteric coating, theenteric coating being designed to protect and release the pharmaceuticalcomposition in a controlled manner within the lower gastrointestinalsystem of a subject, and to avoid systemic side effects. In addition toenteric coatings, the peptide molecules of the instant invention may beencapsulated, coated, engaged or otherwise associated within anycompatible oral drug delivery system or component. For example, in someembodiments a peptide molecule of the present invention is provided in alipid carrier system comprising at least one of polymeric hydrogels,nanoparticles, microspheres, micelles, and other lipid systems.

To overcome peptide degradation in the small intestine, someimplementations of the present invention comprise a hydrogel polymercarrier system in which a peptide molecule in accordance with thepresent invention is contained, whereby the hydrogel polymer protect thepeptide from proteolysis in the small intestine. The peptide moleculesof the present invention may further be formulated for compatible usewith a carrier system that is designed to increase the dissolutionkinetics and enhance intestinal absorption of the peptides. Thesemethods include the use of liposomes, micelles and nanoparticles toincrease GI tract permeation of peptides.

Various bioresponsive systems may also be combined with one or morethioether peptide molecules of the present invention to provide apharmaceutical agent for oral delivery. In some embodiments, a peptidemolecule of the instant invention is used in combination with abioresponsive system, such as hydrogels and mucoadhesive polymers withhydrogen bonding groups (e.g., PEG, poly(methacrylic) acid [PMAA],cellulose, Eudragit®, chitosan and alginate) to provide a therapeuticagent for oral administration. Other embodiments include a method foroptimizing or prolonging drug residence time for a peptide moleculedisclosed herein, wherein the surface of the peptide molecule ismodified to comprise mucoadhesive properties through hydrogen bonds,polymers with linked mucins or/and hydrophobic interactions. Thesemodified peptide molecules may demonstrate increase drug residence timewithin the subject, in accordance with a desired feature of theinvention. Moreover, targeted mucoadhesive systems may specifically bindto receptors at the enterocytes and M-cell surfaces, thereby furtherincreasing the uptake of particles containing the peptide molecules.

Other embodiments comprise a method for oral delivery of a thioetherpeptide molecule described herein, e.g., corresponding to any of SEQ IDNOs: 1-384 or Tables 4 or 5, wherein the peptide molecule is used incombination with permeation enhancers that promote the transport of thepeptides across the intestinal mucosa by increasing paracellular ortranscellular permeation. For example, in one embodiment a permeationenhancer is combined with a thioether peptide molecule described herein,e.g., corresponding to any of SEQ ID NOs: 1-384, or Tables 4 or 5,wherein the permeation enhancer comprises at least one of a long-chainfatty acid, a bile salt, an amphiphilic surfactant, and a chelatingagent. In one embodiment, a permeation enhancer comprising sodiumN-[(hydroxybenzoyl)amino]caprylate is used to form a weak noncovalentassociation with the peptide molecule of the instant invention, whereinthe permeation enhancer favors membrane transport and furtherdissociation once reaching the blood circulation. In another embodiment,a peptide molecule of the present invention is conjugated tooligoarginine, thereby increasing cellular penetration of the peptideinto various cell types. Further, in at least one embodiment anoncovalent bond is provided between a thioether peptide moleculedescribed herein, e.g., SEQ ID NOs: 1-384 or Tables 4 or 5, and apermeation enhancer selected from the group consisting of a cyclodextrin(CD) and a dendrimers, wherein the permeation enhancer reduces peptideaggregation and increasing stability and solubility for the peptidemolecule.

Other embodiments of the invention provide a method for treating anindividual with an α4β7 integrin antagonist thioether peptide moleculehaving an increased half-life. In one aspect, the present inventionprovides an integrin antagonist thioether peptide molecule having ahalf-life of at least several hours to one day in vitro or in vivo(e.g., when administered to a human subject) sufficient for daily (q.d.)or twice daily (b.i.d.) dosing of a therapeutically effective amount. Inanother embodiment, the peptide molecule has a half-life of three daysor longer sufficient for weekly (q.w.) dosing of a therapeuticallyeffective amount. Further, in another embodiment the peptide moleculehas a half-life of eight days or longer sufficient for bi-weekly(b.i.w.) or monthly dosing of a therapeutically effective amount. Inanother embodiment, the thioether peptide molecule is derivatized ormodified such that is has a longer half-life as compared to anunderivatized or unmodified peptide molecule. In another embodiment, thepeptide molecule contains one or more chemical modifications to increaseserum half-life.

When used in at least one of the treatments or delivery systemsdescribed herein, a therapeutically effective amount of one of thethioether peptide molecules of the present invention may be employed inpure form or, where such forms exist, in pharmaceutically acceptablesalt form. As used herein, a “therapeutically effective amount” of thecompound of the invention is meant to describe a sufficient amount ofthe thioether peptide molecule to treat an integrin-related disease,(for example, to reduce inflammation associated with IBD) at a desiredbenefit/risk ratio applicable to any medical treatment. It will beunderstood, however, that the total daily usage of the compounds andcompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular patient willdepend upon a variety of factors including: a) the disorder beingtreated and the severity of the disorder; b) activity of the specificcompound employed; c) the specific composition employed, the age, bodyweight, general health, sex and diet of the patient; d) the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; e) the duration of the treatment; f) drugsused in combination or coincidental with the specific compound employed,and like factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of the compound at levelslower than those required to achieve the desired therapeutic effect andto gradually increase the dosage until the desired effect is achieved.

Alternatively, a compound of the present invention may be administeredas pharmaceutical compositions containing the thioether peptide moleculeof interest in combination with one or more pharmaceutically acceptableexcipients. A pharmaceutically acceptable carrier or excipient refers toa non-toxic solid, semi-solid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The compositions may beadministered parenterally, intracistemally, intravaginally,intraperitoneally, intrarectally, topically (as by powders, ointments,drops, suppository, or transdermal patch), rectally, or buccally. Theterm “parenteral” as used herein refers to modes of administration whichinclude intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous, intradermal and intraarticular injection and infusion.

In particular embodiments, pharmaceutical compositions for parenteralinjection comprise pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), carboxymethylcellulose and suitable mixturesthereof, vegetable oils (such as olive oil), and injectable organicesters such as ethyl oleate. Proper fluidity may be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

These compositions may also contain adjuvants such as preservative,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. Prolonged absorption of the injectable pharmaceutical formmay be brought about by the inclusion of agents which delay absorption,such as aluminum monostearate and gelatin.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide,poly(orthoesters), poly(anhydrides), and (poly)glycols, such as PEG.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Depot injectable formulations are also prepared by entrapping the drugin liposomes or microemulsions which are compatible with body tissues.

The injectable formulations may be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Topical administration includes administration to the skin or mucosa,including surfaces of the lung and eye. Compositions for topical lungadministration, including those for inhalation and intranasal, mayinvolve solutions and suspensions in aqueous and non-aqueousformulations and can be prepared as a dry powder which may bepressurized or non-pressurized. In non-pressurized powder compositions,the active ingredient in finely divided form may be used in admixturewith a larger-sized pharmaceutically acceptable inert carrier comprisingparticles having a size, for example, of up to 100 micrometers indiameter. Suitable inert carriers include sugars such as lactose.

Alternatively, the composition may be pressurized and contain acompressed gas, such as nitrogen or a liquefied gas propellant. Theliquefied propellant medium and indeed the total composition ispreferably such that the active ingredient does not dissolve therein toany substantial extent. The pressurized composition may also contain asurface active agent, such as a liquid or solid non-ionic surface activeagent or may be a solid anionic surface active agent. It is preferred touse the solid anionic surface active agent in the form of a sodium salt.

A further form of topical administration is to the eye. A compound ofthe invention is delivered in a pharmaceutically acceptable ophthalmicvehicle, such that the compound is maintained in contact with the ocularsurface for a sufficient time period to allow the compound to penetratethe corneal and internal regions of the eye, as for example the anteriorchamber, posterior chamber, vitreous body, aqueous humor, vitreoushumor, cornea, iris/ciliary, lens, choroid/retina and sclera. Thepharmaceutically acceptable ophthalmic vehicle may, for example, be anointment, vegetable oil or an encapsulating material. Alternatively, thecompounds of the invention may be injected directly into the vitreousand aqueous humour.

Compositions for rectal or vaginal administration are preferablysuppositories which may be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat room temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the active compound.

Compounds of the present invention may also be administered in the formof liposomes. As is known in the art, liposomes are generally derivedfrom phospholipids or other lipid substances. Liposomes are formed bymono- or multi-lamellar hydrated liquid crystals that are dispersed inan aqueous medium. Any non-toxic, physiologically acceptable andmetabolizable lipid capable of forming liposomes can be used. Thepresent compositions in liposome form can contain, in addition to acompound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art.

Total daily dose of the compositions of the invention to be administeredto a human or other mammal host in single or divided doses may be inamounts, for example, from 0.0001 to 300 mg/kg body weight daily andmore usually 1 to 300 mg/kg body weight.

Non-Invasive Detection of Intestinal Inflammation

The thioether peptides of the invention may be used for detection,assessment and diagnosis of intestinal inflammation by microPET imagingusing an orally stable thioether peptide monomer or dimer moleculeselected from and corresponding to SEQ ID NOs: 1-32, or described hereinor in the accompanying Figures, and that is further labeled with atleast one of a chelating group and a detectable label as part of anon-invasive diagnostic procedure. In one embodiment, an integrinantagonist thioether peptide monomer or dimer molecule is conjugatedwith a bifunctional chelator to provide an orally stable peptidemolecule. In another embodiment, an integrin antagonist peptide monomeror dimer molecule is radiolabeled to provide an orally stable peptidemolecule. The orally stable, chelated or radiolabeled peptide moleculeis then administered to a subject orally or rectally. In one embodiment,the orally stable peptide monomer or dimer molecule is included indrinking water. Following uptake of the peptide molecules, microPETimaging may be used to visualize inflammation throughout the subject'sbowels and digestive track.

EXAMPLES Example 1 Synthesis of Thioether Peptide Monomer and DimerMolecules

The peptide monomers or peptide subunits of the present invention may besynthesized by many techniques that are known to those skilled in theart. Novel and unique thioether peptide molecules were synthesized andpurified, and dimerized in the case of peptide dimer molecules, usingthe techniques provided herein.

Synthesis

The peptides of the present invention were synthesized using theMerrifield solid phase synthesis techniques on Protein Technology'sSymphony multiple channel synthesizer. The peptides were assembled usingHBTU(O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate),Diisopropylethylamine (DIEA) coupling conditions. Rink Amide MBHA resin(100-200 mesh, 0.57 mmol/g) was used for peptides with C-terminal amidesand pre-loaded Wang Resin with N-a-Fmoc protected amino acid was usedfor peptides with C-terminal acids. The coupling reagents (HBTU and DIEApremixed) were prepared at 100 mmol concentration. Similarly amino acidssolutions were prepared at 100 mmol concentration. The peptides wereassembled using standard Symphony protocols.

Assembly

The peptide sequences were assembled as follows: Resin (250 mg, 0.14mmol) in each reaction vial was washed twice with 4 ml of DMF followedby treatment with 2.5 ml of 20% 4-methyl piperidine (Fmoc de-protection)for 10 min. The resin was then filtered and washed two times with DMF (4ml) and re-treated with N-methyl piperidine for additional 30 minute.The resin was again washed three times with DMF (4 ml) followed byaddition 2.5 ml of amino acid and 2.5 ml of HBTU-DIEA mixture. After 45min of frequent agitations, the resin was filtered and washed threetimed with DMF (4 ml each). For a typical peptide of the presentinvention, double couplings were performed. For N-Me-Arg and2-(Chloromethyl)benzoic acid coupling, double coupling of 2.0 eq2-(Chloromethyl)benzoic acid, 2.0 eq PyAOP, and 4 eq DIEA in DMF for 1hr. Reaction completion was monitored using the Chloranil test. Aftercompleting the coupling reaction, the resin was washed three times withDMF (4 ml each) before proceeding to the next amino acid coupling.

Cleavage

Following completion of the peptide assembly, the peptide was cleavedfrom the resin by treatment with cleavage reagent, TFA:water:TIPS(92.5v:5v:2.5v). The cleavage reagent was able to successfully cleavethe peptide from the resin, as well as all remaining side chainprotecting groups.

The cleavage reaction mixture was stirred for 2 h at room temperature.The spent resin was filtered off. The filtrate was then precipitatedinto cold ethyl ether and centrifuged to collect the peptide. The ethylether was decanted, and the solid precipitate was washed two times withcold ethyl ether. The crude peptide was dissolved in a solution ofacetonitrile:water (7:3 with 1% TFA) and filtered. The quality of linearpeptide was then verified using electrospray ionization massspectrometry (ESI-MS) (Micromass/Waters ZQ) before being purified.

Thioether Bond Formation

The unpurified linear monomer (50 mg) was dissolved in 50:50 ACN:water(2.5 mg/ml) then diluted to about 1 mg/mL in 0.1M Tris-HCl pH8.5 buffer.The reaction was monitored using LCMS. When the reaction is completed(usually overnight), diluted the reaction mixture with water and purifyby RP-HPLC.

Purification

Analytical reverse-phase, high performance liquid chromatography (HPLC)was performed on a Gemini C18 column (4.6 mm×250 mm) (Phenomenex).Semi-Preparative reverse phase HPLC was performed on a Gemini 10 μm C18column (22 mm×250 mm) (Phenomenex) or Jupiter 10 μm, 300 A° C.18 column(21.2 mm×250 mm) (Phenomenex). Separations were achieved using lineargradients of buffer B in A (Mobile phase A: water containing 0.15% TFA,mobile phase B: Acetonitrile (ACN) containing 0.1% TFA), at a flow rateof 1 mL/min (analytical) and 15 mL/min (preparative). Separations wereachieved using linear gradients of buffer B in A (Mobile phase A: watercontaining 0.15% TFA, mobile phase B: Acetonitrile (ACN) containing 0.1%TFA), at a flow rate of 1 mL/min (analytical) and 15 mL/min(preparative).

Linker Activation and Dimerization

Small Scale DIG Linker Activation Procedure:

5 mL of NMP was added to a glass vial containing IDA diacid (304.2 mg, 1mmol), N-hydroxysuccinimide (NHS, 253.2 mg, 2.2 eq. 2.2 mmol) and astirring bar. The mixture was stirred at room temperature to completelydissolve the solid starting materials. N, N′-Dicyclohexylcarbodiimide(DCC, 453.9 mg, 2.2 eq., 2.2 mmol) was then added to the mixture.Precipitation appeared within 10 min and the reaction mixture wasfurther stirred at room temperature overnight. The reaction mixture wasthen filtered to remove the precipitated dicyclohexylurea (DCU). Theactivated linker was kept in a closed vial prior to use fordimerization. The nominal concentration of the activated linker wasapproximately 0.20 M.

For dimerization using PEG linkers, there was no pre-activation stepinvolved. Commercially available pre-activated bi-functional PEG linkerswere used.

Dimerization Procedure:

2 mL of anhydrous DMF was added to a vial containing peptide monomer(0.1 mmol). The pH of the peptide was then adjusted to 8˜9 with DIEA.Activated linker (IDA or PEG13, PEG 25) (0.48 eq relative to monomer,0.048 mmol) was then added to the monomer solution. The reaction mixturewas stirred at room temperature for one hour. Completion of thedimerization reaction was monitored using analytical HPLC. The time forcompletion of dimerization reaction varied depending upon the linker.After completion of reaction, the peptide was precipitated in cold etherand centrifuged. The supernatant ether layer was discarded. Theprecipitation step was repeated twice. The crude dimer was then purifiedusing reverse phase HPLC (Luna C18 support, 10 u, 100 A, Mobile phase A:water containing 0.1% TFA, mobile phase B: Acetonitrile (ACN) containing0.1% TFA, gradient of 15% B and change to 45% B over 60 min, flow rate15 ml/min). Fractions containing pure product were then freeze-dried ona lyophilyzer.

The peptide monomers and peptide dimers shown in Tables 4 and 5 weresynthesized and further characterized. Table 4 shows various monomerpeptide compounds according to various non-limiting representativeembodiments of the present invention. The amino acid residues arenumbers Xaa¹⁻¹⁰, in accordance with Formula (II). However, theseresidues should be understood to also correspond to Xaa⁴⁻¹³ in Formula(I). The amino acid sequence of the peptide is shown, wherein “2-benzyl”indicates 2-methylbenzoyl, and lower case letters indicate D-aminoacids. Each peptide is cyclized via an intramolecular thioether bondbetween the amino acid residue or moiety shown at position 1 and theamino acid residue shown at position 7. Table 5 shows various peptidedimer compounds according to various non-limiting representativeembodiments of the present invention. The amino acid sequence of thepeptide is shown, wherein “2-benzyl” indicates 2-methylbenzoyl, andlower case letters indicate D-amino acids. The amino acid residues arenumbers Xaa¹⁻¹⁰, in accordance with Formula (II). However, theseresidues should be understood to also correspond to Xaa⁴⁻¹³ in Formula(I). Each monomer subunit of the peptide dimer is cyclized via anintramolecular thioether bond between the amino acid residue or moietyshown at position 1 and the amino acid residue shown at position 7. Thepeptide monomer subunits of the peptide dimers are dimerized at theirC-termini by the indicated DIG, ADA, IDA, IDA-Palm, IDA-Lauryl,IDA-oleoyl, or IDA-PEG linker.

TABLE 4 Illustrative Thioether Monomers SEQ Peptide ID NO sequence 1 2 34 5 6 7 8 9 10 391 (thioether) Acetyl N—Me—R S D T L C W k NH2 392(thioether) Acetyl N—Me—R S D T L homoCys W k NH2 51 (thioether)Propionyl N—Me—R S D T L C W k NH2 52 (thioether) alpha- N—Me—R S D T LC W k NH2 bromoispbutyryl 53 (thioether) Acetyl N—Me—R S D T L Pen W kNH2 54 (thioether) Propionyl N—Me—R S D T L Pen W k NH2 55 (thioether)2-Benzyl N—Me—R S D T L C W E k NH2 56 (thioether) 2-Benzyl N—Me—R S D TL Pen W E k NH2 57 (thioether) Propionyl N—Me—R S D T L hC W k NH2 58((thioether) Butyryl N—Me—R S D T L C W k NH2)2 59 (thioether) 2-BenzylR S D T L C W k NH2 60 (thioether) 2-Benzyl N—Me—R S D T L Pen W e k NH261 (thioether) 2-Benzyl N—Me—R S D T L Pen W b-H-E k NH2 62 (thioether)2-Benzyl N—Me—R S D T L Pen W E N—Me—k NH2 63 (thioether) 2-BenzylN—Me—R S D T L Pen W Y N—Me—K NH2 64 (thioether) 2-Benzyl N—Me—R S D TNle Pen W E k NH2 65 (thioether) 2-Benzyl N—Me—R S D T L Pen F e k NH266 (thioether) 2-Benzyl N—Me—R S D T L c W b-H-E k NH2 67 (thioether)2-Benzyl N—Me—R S D T L Hcys W E k NH2 68 (thioether) 2-Benzyl N—Me—R SD T L Pen 1-Nal e k NH2 69 (thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nale N—Me—K NH2 70 (thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal b-H-E kNH2 71 (thioether) 2-Benzyl N—Me—R S D T L Pen f 2-Nal k NH2 72(thioether) 2-Benzyl N—Me—R S D T L Pen f E k NH2 73 (thioether)2-Benzyl N—Me—R S D T L Pen F b-H-E k NH2 74 (thioether) 2-Benzyl N—Me—RS D T L Pen Y b-H-E k NH2 75 (thioether) 2-Benzyl N—Me—R S D T L Pen2-Nal e k NH2 76 (thioether) 2-Benzyl N—Me—R S D T L Pen F(CF3) E k NH277 (thioether) 2-Benzyl N—Me—R S D T L Pen 1Nal E k NH2 78 (thioether)2-Benzyl N—Me—R S D T L Pen Y E k NH2 79 (thioether) 2-Benzyl N—Me—R S DT L Pen Y e k NH2 80 (thioether) 2-Benzyl N—Me—R S D T L Pen W E k(Ac)NH2 81 (thioether) 2-Benzyl N—Me—R S D T L Pen W e k(Ac) NH2 82(thioether) 2-Benzyl N—Me—R S D T L Pen W e k(PEG8) NH2 83 (thioether)2-Benzyl N—Me—R S D T L Pen W b-H-E k(Ac) NH2 84 (thioether) 2-BenzylN—Me—R S D T L Pen W E N—Me—k(Ac) NH2 85 (thioether) 2-Benzyl N—Me—R S DT L Pen W Y N—Me—K(Ac) NH2 86 (thioether) 2-Benzyl N—Me—R S D T Nle PenW E k(Ac) NH2 87 (thioether) 2-Benzyl N—Me—R S D T L Pen F e k(Ac) NH288 (thioether) 2-Benzyl N—Me—R S D T L Pen F(CF3) E k(Ac) NH2 89(thioether) 2-Benzyl N—Me—R S D T L Pen 1Nal E k(Ac) NH2 90 (thioether)2-Benzyl N—Me—R S D T L Pen Y E k(Ac) NH2 91 (thioether) 2-Benzyl N—Me—RS D T L Pen Y e k(Ac) NH2 92 ((thioether) 2-Benzyl N—Me—R S D T L Pen WE Dap NH2 93 ((thioether) 2-Benzyl N—Me—R S D T L Pen W E Dab NH2 94((thioether) 2-Benzyl N—Me—R S D T L Pen W e Dap NH2 95 ((thioether)2-Benzyl N—Me—R S D T L Pen W e Dab NH2 96 ((thioether) 2-Benzyl N—Me—RS D T L Pen W E NH2 97 ((thioether) 2-Benzyl N—Me—R S D T L Pen W e NH298 ((thioether) 2-Benzyl N—Me—R S D T L Pen W e NH2 99 ((thioether)2-Benzyl N—Me—R S D T L Pen W e L NH2 100 ((thioether) 2-Benzyl N—Me—R SD T L Pen W e S NH2 101 ((thioether) 2-Benzyl N—Me—R S D T L Pen W e FNH2 102 ((thioether) 2-Benzyl N—Me—R S D T L Pen W e H NH2 103((thioether) 2-Benzyl N—Me—R S D T L Pen W e Q NH2 104 ((thioether)2-Benzyl N—Me—R S D T L Pen W e Y NH2 105 ((thioether) 2-Benzyl N—Me—R SD T L Pen W e I NH2 106 ((thioether) 2-Benzyl N—Me—R S D T L Pen W e sNH2 107 ((thioether) 2-Benzyl N—Me—R S D T L Pen W e f NH2 108((thioether) 2-Benzyl N—Me—R S D T L Pen W e e NH2 109 ((thioether)2-Benzyl N—Me—R S D T L Pen W e h NH2 110 ((thioether) 2-Benzyl N—Me—R SD T L Pen W e y NH2 111 ((thioether) 3-Benzyl N—Me—R S D T L Pen W e kNH2 112 (thioether) 4-Benzyl N—Me—R S D T L Pen W e k NH2 113((thioether) 2-Benzyl N—Me—R S D T L Pen W e E NH2 114 ((thioether)2-Benzyl N—Me—R S D T L Pen 2-Nal e NH2 115 ((thioether) 2-Benzyl N—Me—RS D T L Pen W E(OMe) k NH2 116 ((thioether) 2-Benzyl N—Me—R S D T L Pen2-Nal NH2 117 (thioether) 2-Benzyl N—Me—R S D T L C Tic E k NH2 118(thioether) 2-Benzyl N—Me—R S D T L C Tic k OH 119 ((thioether) 2-BenzylN—Me—R S D T L Pen Atc bHE NH2 120 ((thioether) 2-Benzyl N—Me—R S D T LPen erythro-b-F—S bHE NH2 121 ((thioether) 2-Benzyl N—Me—R S D T L Penerythro-b-F—S bHE NH2 122 ((thioether) 2-Benzyl N—Me—R S D T L Penthreo-b-F—S bHE NH2 123 ((thioether) 2-Benzyl N—Me—R S D T L Penthreo-b-F—S bHE NH2 124 ((thioether) 2-Benzyl N—Me—R S D T L Pen Bpa bHENH2 125 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(3-Me) bHE NH2 126((thioether) 2-Benzyl N—Me—R S D T L Pen F(2-Me) bHE NH2 127((thioether) 2-Benzyl N—Me—R S D T L Pen F(2-CF3)) bHE NH2 128((thioether) 2-Benzyl N—Me—R S D T L Pen b-Me—F bHE NH2 129 ((thioether)2-Benzyl N—Me—R S D T L Pen b-Me—F bHE NH2 130 ((thioether) 2-BenzylN—Me—R S D T L Pen b-dimethyl-F bHE NH2 131 ((thioether) 2-Benzyl N—Me—RS D T L Pen b-dimethyl-F bHE NH2 132 ((thioether) 2-Benzyl N—Me—R S D TL Pen 4-Me—F bHE NH2 133 ((thioether) 2-Benzyl N—Me—R S D T L Pen BipbHE NH2 134 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-tBu) b-H-E NH2135 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) N—Me—E NH2 136((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) N—Me—D NH2 137((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) alpha-H-E NH2 138((thioether) 2-Benzyl Cit S D T L Pen F(4-tBu) b-H-E NH2 139((thioether) 2-Benzyl N—Me—R A D T L Pen F(4-tBu) b-H-E NH2 140((thioether) 2-Benzyl N—Me—R Abu D T L Pen F(4-tBu) b-H-E NH2 141((thioether) 2-Benzyl N—Me—R Tbu D T L Pen F(4-tBu) b-H-E NH2 142((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-tBu) N—Me—E OH 224thioether 2-Benzyl N—Me—R S D T L Pen W e Dap Ac 225 thioether 2-BenzylN—Me—R S D T Nle Pen F e N—Me—k NH2 226 thioether 2-Benzyl N—Me—R S D TNle Pen W E N—Me—K NH2 227 thioether 2-Benzyl N—Me—R S D T Nle Pen F eN—Me—k NH2 228 thioether 2-Benzyl N—Me—R S D T Nle Pen W E N—Me—k NH2229 thioether 2-Benzyl N—Me—R S D T Nle Pen F e N—Me—k NH2 230 AcC(thioether N—Me—R S D T L C(thioether W E k NH2 propane) propane) 231thioether 2-Benzyl N—Me—R S D T L Pen W E Dab Ac 232 thioether 2-BenzylN—Me—R S D T L Pen W e Dab Ac 233 ((thioether) 2-Benzyl N—Me—R S D T LPen W E Dab NH2 234 thioether 2-Benzyl N—Me—R S D T L Pen W E Dap Ac 235((thioether) 2-Benzyl N—Me—R S D T L C Tic e k NH2 236 ((thioether)2-Benzyl N—Me—R S D T L Pen W f k NH2 237 ((thioether) 2-Benzyl N—Me—R SD T L Pen W y k NH2 238 ((thioether) 2-Benzyl N—Me—R S D T L C Tic e kNH2 239 ((thioether) 2-Benzyl N—Me—R S D T L Pen W P k NH2 240((thioether) 2-Benzyl N—Me—R S D T L Pen W P K NH2 241 ((thioether)2-Benzyl N—Me—R S D T L Pen W p K NH2 242 ((thioether) 2-Benzyl N—Me—R SD T L Pen F(2- e k NH2 carbamoyl) 243 ((thioether) 2-Benzyl N—Me—R S D TL Pen F(3- e k NH2 carbamoyl) 244 ((thioether) 2-Benzyl N—Me—R S D T LPen F(4-COOH) e k NH2 245 ((thioether) 2-Benzyl N—Me—R S D T L PenF(2,4-Cl) e k NH2 246 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(3,4-Cl)e k NH2 247 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-OMe) e k NH2248 ((thioether) 2-Benzyl N—Me—R S D T L Pen W h k NH2 249 ((thioether)2-Benzyl N—Me—R S D T L Pen W F(4- k NH2 COOH) 250 ((thioether) 2-BenzylN—Me—R S D T L Pen F(4tBu) e k NH2 251 ((thioether) 2-Benzyl N—Me—R S DT L Pen F(4-F) e k NH2 252 ((thioether) 2-Benzyl N—Me—R S D T L Pen Bipe k NH2 253 ((thioether) 2-Benzyl N—Me—R S D T L Pen W Tic k NH2 254((thioether) 2-Benzyl N—Me—R S D T L Pen W w k NH2 255 ((thioether)2-Benzyl N—Me—R S D T L Pen 1-Nal f k NH2 256 ((thioether) 2-BenzylN—Me—R S D T L Pen 1-Nal h k NH2 257 ((thioether) 2-Benzyl N—Me—R S D TL Pen 1-Nal l k NH2 258 ((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal rk NH2 259 ((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal Tic k NH2 260((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal t k NH2 261 ((thioether)2-Benzyl N—Me—R S D T L Pen 2-Nal f k NH2 262 ((thioether) 2-BenzylN—Me—R S D T L Pen 2-Nal h k NH2 263 ((thioether) 2-Benzyl N—Me—R S D TL Pen 2-Nal l k NH2 264 ((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal rk NH2 265 ((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal Tic k NH2 266((thioether) 2-Benzyl N—Me—R S D T L Pen F(4CF3) e k NH2 267((thioether) 2-Benzyl N—Me—R S D T L Pen Y e k NH2 268 ((thioether)2-Benzyl N—Me—R S D T L Pen H e k NH2 269 ((thioether) 2-Benzyl N—Me—R SD T L Pen F(4tBu) E k NH2 270 ((thioether) 2-Benzyl N—Me—R S D T L PenF(4tBu) b- k NH2 HomoGlu 271 ((thioether) 2-Benzyl N—Me—R S D T L PenF(4-COOH) E k NH2 272 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-COOH)E k NH2 273 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-COOH) E k NH2274 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-COOH) b- k NH2 HomoGlu275 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-COOH) b- k NH2 HomoGlu276 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-COOH) b- k NH2 HomoGlu277 ((thioether) 2-Benzyl N—Me—R S D T L Pen Bip E k NH2 278((thioether) 2-Benzyl N—Me—R S D T L Pen Bip b- k NH2 HomoGlu 279((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal E k NH2 280 ((thioether)2-Benzyl N—Me—R S D T L Pen 2-Nal b- k NH2 HomoGlu 281 ((thioether)2-Benzyl N—Me—R S D T L Pen 1-Nal E k NH2 282 ((thioether) 2-BenzylN—Me—R S D T L Pen 1-Nal b- k NH2 HomoGlu 283 ((thioether) 2-BenzylN—Me—R S D T L Pen 2-Nal k NH2 284 ((thioether) 2-Benzyl N—Me—R S D T LPen 2-Nal k NH2 285 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) EN—Me—K NH2 286 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) E N—Me—kNH2 287 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) b-Homo N—Me—KNH2 Glu 288 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) b-HomoN—Me—k NH2 Glu 289 ((thioether) 2-Benzyl N—Me—R S D T L Pen Bip E N—Me—KNH2 290 ((thioether) 2-Benzyl N—Me—R S D T L Pen Bip E N—Me—k NH2 291((thioether) 2-Benzyl N—Me—R S D T L Pen Bip b-Homo N—Me—K NH2 Glu 292((thioether) 2-Benzyl N—Me—R S D T L Pen Bip b-Homo N—Me—k NH2 Glu 293((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal E N—Me—K NH2 294((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal E N—Me—k NH2 295((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal b- N—Me—K NH2 HomoGlu 296((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal b-Homo N—Me—k NH2 Glu 297((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal E N—Me—K NH2 298((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal E N—Me—k NH2 299((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal b-Homo N—Me—K NH2 Glu 300((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal b-Homo N—Me—k NH2 Glu

TABLE 5 Illustrative Thioether Dimers SEQ ID NO Peptide sequence 1 2 3 45 6 7 8 9 10 Linker 143 [(thioether) Acetyl N—Me—R S D T L C W k NH2]2DIG 144 [(thioether) Propionyl N—Me—R S D T L C W k NH2]2 DIG 145[(thioether) 2-Benzyl N—Me—R S D T L C W E k NH2]2 DIG 146 [(thioether)2-Benzyl N—Me—R S D T L Pen W E k NH2]2 DIG 147 ((thioether) AcetylN—Me—R S D T L Pen W k NH2)2 DIG 148 ((thioether) Propionyl N—Me—R S D TL Pen W k NH2)2 DIG 149 [(thioether) Propionyl N—Me—R S D T L hC W kNH2]2 DIG 150 ((thioether) 2-Benzyl N—Me—R S D T L Pen W e k NH2)2 DIG151 ((thioether) 2-Benzyl N—Me—R S D T L Pen W b-H-E k NH2)2 DIG 152((thioether) 2-Benzyl N—Me—R S D T L Pen W E N—Me—k NH2)2 DIG 153((thioether) 2-Benzyl N—Me—R S D T L Pen W Y N—Me—K NH2)2 DIG 154((thioether) 2-Benzyl N—Me—R S D T Nle Pen W E k NH2)2 DIG 155((thioether) 2-Benzyl N—Me—R S D T L Pen F e k NH2)2 DIG 156 (thioether)2-Benzyl N—Me—R S D T L c W b-H-E k NH2)2 DIG 157 (thioether) 2-BenzylN—Me—R S D T L Hcys W E k NH2)2 DIG 158 (thioether) 2-Benzyl N—Me—R S DT L Pen 1-Nal e k NH2)2 DIG 159 (thioether) 2-Benzyl N—Me—R S D T L Pen2-Nal e k NH2)2 DIG 160 (thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal eN—Me—K NH2)2 DIG 161 (thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal b-H-Ek NH2)2 DIG 162 (thioether) 2-Benzyl N—Me—R S D T L Pen f 2-Nal k NH2)2DIG 163 (thioether) 2-Benzyl N—Me—R S D T L Pen f E k NH2)2 DIG 164(thioether) 2-Benzyl N—Me—R S D T L Pen F b-H-E k NH2)2 DIG 165(thioether) 2-Benzyl N—Me—R S D T L Pen Y b-H-E k NH2)2 DIG 166((thioether) 2-Benzyl N—Me—R S D T L Pen F(CF3) E k NH2)2 DIG 167((thioether) 2-Benzyl N—Me—R S D T L Pen 1Nal E k NH2)2 DIG 168((thioether) 2-Benzyl N—Me—R S D T L Pen Y E k NH2)2 DIG 169((thioether) 2-Benzyl N—Me—R S D T L Pen Y e k NH2)2 DIG 170((thioether) 2-Benzyl N—Me—R S D T L Pen W e k NH2)2 ADA 171((thioether) 2-Benzyl N—Me—R S D T L Pen W e k NH2)2 IDA 172 (thioether)2-Benzyl N—Me—R S D T L Pen(═O) 2-Nal e k NH2)2 DIG 173 (thioether)2-Benzyl N—Me—R S D T L Pen(═O) 2-Nal e k NH2)2 DIG 174 ((thioether)2-Benzyl N—Me—R S D T L Pen W e k NH2)2 IDA-Biotine 175 ((thioether)2-Benzyl N—Me—R S D T L Pen W e k NH2)2 IDA-PEG4-Biotin 176 ((thioether)2-Benzyl N—Me—R S D T L Pen F(2,4-diCl) e k NH2)2 DIG 177 ((thioether)2-Benzyl N—Me—R S D T L Pen F(3,4-diCl) e k NH2)2 DIG 178 ((thioether)2-Benzyl N—Me—R S D T L Pen Bip e k NH2)2 DIG 179 ((thioether) 2-BenzylN—Me—R S D T L c Aic e k NH2)2 DIG 180 ((thioether) 2-Benzyl N—Me—R S DT L C Aic e k NH2)2 DIG 181 ((thioether) 2-Benzyl N—Me—R S D T L D-Pen WE k NH2)2 DIG 182 ((thioether) 2-Benzyl N—Me—R S D T L C N—Me—Y E kNH2)2 DIG 183 ((thioether) 2-Benzyl N—Me—R S D T L C N—Me—F E k NH2)2DIG 184 ((thioether) 2-Benzyl N—Me—R S D T L C Tic e k NH2)2 DIG 185((thioether) 2-Benzyl N—Me—R S D T L c Tic e k NH2)2 DIG 186((thioether) 2-Benzyl N—Me—R S D T L C f E k NH2)2 DIG 187 ((thioether)2-Benzyl N—Me—R S D T L C f e k NH2)2 DIG 188 ((thioether) 2-BenzylN—Me—R S D T L D-Pen Y e k NH2)2 DIG 189 ((thioether) 2-Benzyl N—Me—R SE T L Pen F e k NH2)2 DIG 190 ((thioether) 2-Benzyl N—Me—R S D T L Pen We L k NH2)2 DIG 191 ((thioether) 2-Benzyl N—Me—R S D T L Pen W e S kNH2)2 DIG 192 ((thioether) 2-Benzyl N—Me—R S D T L Pen W e F k NH2)2 DIG193 ((thioether) 2-Benzyl N—Me—R S D T L Pen W e H k NH2)2 DIG 194((thioether) 2-Benzyl N—Me—R S D T L Pen W e E k NH2)2 DIG 195((thioether) 2-Benzyl N—Me—R S D T L Pen W e Y k NH2)2 DIG 196((thioether) 2-Benzyl N—Me—R S D T L Pen W e I (D-L) k NH2)2 DIG 197((thioether) 2-Benzyl N—Me—R S D T L Pen W e s k NH2)2 DIG 198((thioether) 2-Benzyl N—Me—R S D T L Pen W e f k NH2)2 DIG 199((thioether) 2-Benzyl N—Me—R S D T L Pen W e h k NH2)2 DIG 200((thioether) 2-Benzyl N—Me—R S D T L Pen W e e k NH2)2 DIG 201((thioether) 2-Benzyl N—Me—R S D T L Pen W e y k NH2)2 DIG 202((thioether) 2-Benzyl N—Me—R S D T L Pen W Bip k NH2)2 DIG 203((thioether) 2-Benzyl N—Me—R S D T L Pen F Bip k NH2)2 DIG 204((thioether) 2-Benzyl N—Me—R S D T L Pen F e k OH)2 DIG 205 ((thioether)2-Benzyl N—Me—R S D T L C Tic Bip k NH2)2 DIG 206 (thioether) 2-BenzylN—Me—R S D T L Pen 2-Nal Bip k NH2)2 DIG 207 ((thioether) 2-BenzylN—Me—R S D T L C Tic e k OH)2 DIG 208 ((thioether) 2-Benzyl N—Me—R S D TL Pen Bip e k OH)2 DIG 209 ((thioether) 2-Benzyl N—Me—R S D T L Pen W ek OH)2 DIG 210 ((thioether) 2-Benzyl N—Me—R S D T L Pen W E(OMe) k NH2DIG 211 ((thioether) 2-Benzyl N—Me—R S D T L C Tic E(OMe) k NH2 DIG 212((thioether) 2-Benzyl N—Me—R S D T L Pen W e k NH2)2 IDA-Palm 213((thioether) 2-Benzyl N—Me—R S D T L Pen W e k NH2)2 IDA-Lauryl 214((thioether) 2-Benzyl N—Me—R S D T L Pen W e k NH2)2 IDA-oleoyl 215((thioether) 2-Benzyl N—Me—R S D T L Pen W e k NH2)2 IDA-PEG12-NH2 216((thioether) 2-Benzyl N—Me—R S D T L C Tic k NH2)2 DIG 217 ((thioether)2-Benzyl N—Me—R S D T L Pen W e k NH2)2 IDA-PEG12-NH-oleoyl 218((thioether) 2-Benzyl N—Me—R S D T L Pen W e k NH2)2 IDA-PEG12-NH-Lauryl219 ((thioether) 2-Benzyl N—Me—R S D T L C Tic E k NH2)2 DIG 220((thioether) 2-Benzyl N—Me—R S D T L C Tic E(OMe) k OH)2 DIG 221((thioether) 2-Benzyl N—Me—R S D T L C Tic k OH)2 DIG 222 ((thioether)2-Benzyl N—Me—R S D T L Pen F(4-tBu) bHE k NH2)2 DIG 223 ((thioether)2-Benzyl N—Me—R S D T L Pen F(4-tBu) bHE k OH)2 DIG 301 (thioetherButyryl N—Me—R S D T L C W k NH2)2 DIG 302 (thioether 2-Benzyl N—Me—R SD T L c W b-H-E k NH2)2 DIG 303 (thioether 2-Benzyl N—Me—R S D T L HcysW E k NH2)2 DIG 304 thioether 2-Benzyl N—Me—R S D T L Pen W E Dap Ac 305thioether 2-Benzyl N—Me—R S D T L Pen W E Dab Ac 306 thioether 2-BenzylN—Me—R S D T L Pen W e Dap Ac 307 thioether 2-Benzyl N—Me—R S D T L PenW e Dab Ac 308 (thioether 2-Benzyl N—Me—R S D T L Pen W e k NH2)2 DIG309 thioether 2-Benzyl N—Me—R S D T L Pen W e NH2 310 (thioether3-Benzyl N—Me—R S D T L Pen W e k NH2)2 DIG 311 (thioether 4-BenzylN—Me—R S D T L Pen W e k NH2)2 DIG 312 ((thioether) 2-Benzyl N—Me—R S DT L Pen W e k NH2)2 IDA-PEG12-NH-Lauryl 313 (thioether) 2-Benzyl N—Me—RS D T L C Tic k OH 314 ((thioether) 2-Benzyl N—Me—R S D T L Pen W E(OMe)k NH2)2 DIG 315 ((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal f k NH2)2DIG 316 ((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal h k NH2)2 DIG 317((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal l k NH2)2 DIG 318((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal r k NH2)2 DIG 319((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal Tic k NH2)2 DIG 320((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal t k NH2)2 DIG 321((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal E k NH2)2 DIG 322((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal b-HomoGlu k NH2)2 DIG 323((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal E N—Me—K NH2)2 DIG 324((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal E N—Me—k NH2)2 DIG 325((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal b-HomoGlu N—Me—K NH2)2DIG 326 ((thioether) 2-Benzyl N—Me—R S D T L Pen 1-Nal b-HomoGlu N—Me—kNH2)2 DIG 327 ((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal f k NH2)2DIG 328 ((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal h k NH2)2 DIG 329((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal l k NH2)2 DIG 330((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal r k NH2)2 DIG 331((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal Tic k NH2)2 DIG 332((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal E k NH2)2 DIG 333((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal b-HomoGlu k NH2)2 DIG 334((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal k NH2 DIG 335((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal k NH2 DIG 336((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal E N—Me—K NH2)2 DIG 337((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal E N—Me—k NH2)2 DIG 338((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal b-HomoGlu N—Me—K NH2)2DIG 339 ((thioether) 2-Benzyl N—Me—R S D T L Pen 2-Nal b-HomoGlu N—Me—kNH2)2 DIG 340 ((thioether) 2-Benzyl N—Me—R S D T L Pen Bip e k NH2)2 DIG341 ((thioether) 2-Benzyl N—Me—R S D T L Pen Bip E k NH2)2 DIG 342((thioether) 2-Benzyl N—Me—R S D T L Pen Bip b-HomoGlu k NH2)2 DIG 343((thioether) 2-Benzyl N—Me—R S D T L Pen Bip E N—Me—K NH2)2 DIG 344((thioether) 2-Benzyl N—Me—R S D T L Pen Bip E N—Me—k NH2)2 DIG 345((thioether) 2-Benzyl N—Me—R S D T L Pen Bip b-HomoGlu N—Me—K NH2)2 DIG346 ((thioether) 2-Benzyl N—Me—R S D T L Pen Bip b-HomoGlu N—Me—k NH2)2DIG 347 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(2,4-Cl) e k NH2)2 DIG348 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(2-carbamoyl) e k NH2)2DIG 349 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(3,4-Cl) e k NH2)2 DIG350 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(3-carbamoyl) e k NH2)2DIG 351 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4CF3) e k NH2)2 DIG352 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-COOH) e k NH2)2 DIG 353((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-COOH) E k NH2)2 DIG 354((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-COOH) E k NH2)2 DIG 355((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-COOH) E k NH2)2 DIG 356((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-COOH) b-HomoGlu k NH2)2 DIG357 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-COOH) b-HomoGlu k NH2)2DIG 358 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-COOH) b-HomoGlu kNH2)2 DIG 359 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-F) e k NH2)2DIG 360 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4-OMe) e k NH2)2 DIG361 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) e k NH2)2 DIG 362((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) E k NH2)2 DIG 363((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) b-HomoGlu k NH2)2 DIG364 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) E N—Me—K NH2)2 DIG365 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) E N—Me—k NH2)2 DIG366 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) b-HomoGlu N—Me—KNH2)2 DIG 367 ((thioether) 2-Benzyl N—Me—R S D T L Pen F(4tBu) b-HomoGluN—Me—k NH2)2 DIG 368 ((thioether) 2-Benzyl N—Me—R S D T L Pen H e kNH2)2 DIG 369 ((thioether) 2-Benzyl N—Me—R S D T L C Tic e k NH2 DIG 370((thioether) 2-Benzyl N—Me—R S D T L C Tic e k NH2)2 DIG 371((thioether) 2-Benzyl N—Me—R S D T L Pen W E Dab NH2)2 DIG 372((thioether) 2-Benzyl N—Me—R S D T L Pen W f k NH2)2 DIG 373((thioether) 2-Benzyl N—Me—R S D T L Pen W y k NH2)2 DIG 374((thioether) 2-Benzyl N—Me—R S D T L Pen W P k NH2)2 DIG 375((thioether) 2-Benzyl N—Me—R S D T L Pen W P K NH2)2 DIG 376((thioether) 2-Benzyl N—Me—R S D T L Pen W p K NH2)2 DIG 377((thioether) 2-Benzyl N—Me—R S D T L Pen W h k NH2)2 DIG 378((thioether) 2-Benzyl N—Me—R S D T L Pen W F(4-COOH) k NH2)2 DIG 379((thioether) 2-Benzyl N—Me—R S D T L Pen W Tic k NH2)2 DIG 380((thioether) 2-Benzyl N—Me—R S D T L Pen W w k NH2)2 DIG 381 thioetherAcetyl N—Me—R S D T L Pen W k NH2)2 DIG 382 thioether Propionyl N—Me—R SD T L Pen W k NH2)2 DIG 383 thioether Propionyl N—Me—R S D T L hC W kNH2)2 DIG 384 ((thioether) 2-Benzyl N—Me—R S D T L Pen Y e k NH2)2 DIG

Example 2 Characterization of Thioether Peptide Monomer and DimerMolecules

The stability, potency, and selectivity of certain thioether peptidemonomer and dimers were determined using a variety of assays describedherein. Peptides listed in Table 8 can be used as control peptides forall of the assays described herein.

Simulated Intestinal Fluid (SIF) Stability Assay

Studies were carried out in simulated intestinal fluid (SIF) to evaluateintestinal stability of the peptide molecules of the instant invention.To prepare the SIF reagent, blank FASSIF was prepared by dissolving0.348 g NaOH, 3.954 g sodium phosphate monobasic monohydrate and 6.186 gNaCl in a final volume of 1 liter water (final pH=6.5). To thissolution, 24 g porcine pancreatin (Sigma catalog P7545) was added andstirred for 30 minutes (final pancreatin concentration is 2.4%). Thesolution was filtered through a cheese cloth and a No. 1 Whatman filter,and 10 ml aliquots were stored at −70° C. To run the reaction, a 10 mlaliquot was thawed at 37° C., and 125 μl aliquots were removed and mixedwith an equal volume of blank FASSIF. The peptide stock solution (10 mMin 100% DMSO) was diluted 75-fold in blank FASSIF. A 50 μl aliquot ofthe diluted peptide was combined with 125 μl pancreatin (2.4%) and 125μl blank FASSIF to yield final concentrations of 1% pancreatin and 22 μMpeptide. The reactions were incubated at 37° C., and at various timepoints 50 μl aliquots were removed and added to 200 μl of quenchsolution containing 50% acetonitrile, 50% methanol, 5% formic acid, and1 μg/ml internal standard. The quenched samples were centrifuged at10,000 rpm for 10 minutes, and the supernatants were analyzed byLCMS/MS. The percent remaining at each time point was calculated basedon the peak area response ratio of test to compound to internalstandard. Half-lives were calculated by fitting to a first-orderexponential decay equation using GraphPad. A small sampling of theresults of these studies is provided and discussed herein and in theaccompanying figures.

Simulated Gastric Fluid (SGF) Stability Assays

Studies were carried out in simulated gastric fluid (SGF) to evaluateintestinal stability of the peptide molecules of the instant invention.SGF was prepared by adding 20 mg NaCl, 32 mg porcine pepsin (MPBiochemicals, catalog 02102599), and 70 μl HCl to 10 ml water (finalpH=2). Aliquots of SGF (0.5 ml each) were pre-warmed at 37° C. To startthe reaction, 1 μl of peptide stock solution (10 mM in DMSO) was addedto 0.5 ml SGF and thoroughly mixed such that the final peptideconcentration was 20 μM. The reactions were incubated at 37° C. withgentle shaking. At each time point (0, 15, 30, 60 min) 50 μl aliquotswere removed and added to 200 ul acetonitrile containing 0.1% formicacid to quench the reaction. Samples are stored at 4° C. until the endof the experiment and centrifuged at 10,000 rpm for 5 minutes. Aliquotsof the supernatant were removed, diluted 1:1 into distilled watercontaining internal standard, and analyzed by LCMS/MS. Percent remainingat each timepoint was calculated based on the peak area response ratioof test to compound to internal standard. Time 0 was set to 100%, andall later timepoints were calculated relative to time 0. Half-lives werecalculated by fitting to a first-order exponential decay equation usingGraphPad.

Redox Stability Assays

Studies were carried out under redox conditions to evaluate intestinalstability of the peptide molecules of the instant invention.

Dithiothreitol (DTT) Redox Stability Assay

The DTT stability assay was prepared by adding 51l of a 10 mM peptidestock solution in DMSO to 1 ml of 100 mM Tris-Cl, pH 7.5 (final peptideconcentration is 50 μM). At time 0 min, 5 ul of a freshly thawed 100 mMDT solution was added such that the final DTT concentration is 0.5 mM.The reactions were incubated at room temperature. At different timepoints up to 120 minutes, 50 μl aliquots were removed and the reactionwas quenched by adding 10l of 5M acetic acid. To measure disappearanceof the parent peptide, the quenched samples (30 μl) were analyzed byreverse phase HPLC and UV absorbance at 220 nm. Half-lives werecalculated by fitting to a first-order exponential decay equation usingExcel.

Cysteine/Cystine Redox Stability Assay

Peptides were diluted to 90 μM by adding 4.545 μl of a 10 mM peptideDMSO stock to 495.45 μl of 100 mM Tris-Cl, pH 7.5. Aliquots of 55 μlwere removed and added to 20 μl of 2.5 mM Cystine in 100 mM Tris-Cl, pH7.5. Cysteine stock solutions in 100 mM Tris-Cl, pH 7.5 were preparedfresh at the following concentrations: 400 mM, 200 mM, 80 mM, 44 mM, 22mM, 1 mM, 5.5 mM and blank. At time 0, 25 μl of each cysteine stocksolution was added to the 55 μl of cystine/peptide solution and themixture was incubated at room temperature for 40 min. The samples werequenched by adding 20l of 5M acetic acid and analyzed by reverse phaseHPLC. The fraction of oxidized peptide was calculated and plottedagainst the calculated oxidation reduction potential (OEP) as defined bythe Nernst equation.

α4β7-MAdCAM Competition ELISA

A nickel coated plate (Pierce #15442) was coated with rh integrin α4β7(R&D Systems #5397-A30) at 800 ng/well and incubated at room temperaturewith shaking for 1 hr. The solution was then removed by shaking andblocked with assay buffer (50 mM Tris-HCl pH7.6, 150 mM NaCl, 1 mM MnCl₂or MgCl₂, 0.05% Tween-20 and 0.5% BSA) at 250 ul/well. The plate wasthen incubated at room temperature for 1 hr. Each well was washed 3times with wash buffer (50 mM Tris-HCl pH7.6, 100 mM NaCl, 1 mM MnCl₂ orMgCl₂, 0.05% Tween-20). To each well was added 25 ul of a serialdilution (3-fold dilutions in assay buffer) of peptides starting at 20μM. 25 ul of recombinant human MAdCAM-1 (R&D Systems #6056-MC) was thenadded to each well at a fixed concentration 20 nM. The final startingpeptide concentration was 10 μM, and the final MAdCAM-1 concentrationwas 10 nM. The plates were then incubated at room temperature for 1 hrto reach binding equilibrium. The wells were then washed three timeswith wash buffer. 50 ul of mouse anti-human IgG1-HRP (Invitrogen #A10648) diluted in 1:2000 in assay buffer was then added to each well.The wells were incubated at room temperature for 45 min with shaking.The wells were then washed 3 times with wash buffer. 100 ul of TMB werethen added to each well and closely observe during development time. Thereaction was stopped with 2N H₂SO₄ and absorbance was read at 450 nm.

α4β1-VCAM Competition ELISA

A Nunc MaxiSorp plate was coated with rh VCAM-1/CD106 Fc chimera (R&D#862-VC) at 400 ng/well in 50 ul per well in 1×PBS and incubatedovernight at 4° C. The solution was removed by shaking and then blockedwith 250 ul of 1% BSA in 1×PBS per well. The wells were then incubatedat room temperature for 1 hr with shaking. Each well was then washedonce with wash buffer (50 mM Tris-HCl pH7.6, 100 mM NaCL, 1 mM MnCl₂ orMgCl₂, 0.05% Tween-20). 25 ul of serial dilutions of peptides startingat 200 μM in assay buffer (Assay buffer: 50 mM Tris-HCl pH7.6, 100 mMNaCl, 1 mM MnCl₂ or MgCl₂, 0.05% Tween-20) was added to each well.Additionally, 25 ul of α4β1 (R&D Systems #5668-A4) was added to eachwell at a fixed concentration of 120 nM. The final peptide and α4β1concentrations were 1001M and 60 nM, respectively. The plates were thenincubated at 37° C. for 2 hr. The solution was then removed by shakingand each well was washed three times with wash buffer. 50 ul of 9F10antibody at 4 ug/ml (purified mouse anti-human CD49d, BD BioscienceCat#555502) was then added to each well, and the plate was incubated atroom temperature for 1 hr with shaking. The solution was again removedby shaking, and each well was washed three times with wash buffer. 50 ulof peroxidase-conjugated AffiniPure Goat anti-mouse IgG (Jackson immuneresearch cat #115-035-003) diluted in 1:5000 in assay buffer was addedto each well. The plate was incubated at room temperature for 30 minwith shaking. Each well was then washed 3 times with wash buffer. 100 ulof TMB was then added to each well and closely observe during developingtime. The reaction was stepped with 2N H₂SO₄ and absorbance was read at450 nm.

PBMC Memory T Cell Adhesion Assay

Fresh CD4+/CD45RO+ memory T cells were isolated from human peripheralblood mononuclear cell (PBMC) donors by Aragen Bioscience Inc. (MorganHill, Calif.). The assay plate was prepared using IgG Fc captureantibody (donkey anti human) immobilized at 500 ng/well in 50 mM sodiumbicarbonate buffer, pH 9.5, ON, 4 C onto a Greiner Fluotrac plate (100ul per well). The plate was rinsed two time with Blocking Buffer (25 mMTris HCl, pH7.5, 150 mM NaCl, 1.5% BSA, 0.05% Tween), and blocked withBlocking Buffer for 2 hours at 37 C or 5 hours at RT using 200 ul perwell. The Blocking Buffer was removed and either MAdCAM-1 or VCAM-1 at400 ng/well in Blocking Buffer was added and the plate incubatedovernight at 4 C (100 ul per well). The plate was washed two times withBlocking Buffer, and rinsed once with 200 ul Binding Media (DMEM phenolred free, 10 mM HEPES, 1×Na pyruvate, 1× Glutamine, and supplementedwith 1 mM MnCl2 prior to use). To prepare cells, approximately 25million CD4+/CD45RO+ memory T cells were counted by trypan blueexclusion using a haemocytometer to determine viability and cell count.The cells were transferred to a 50 ml conical tube, and centrifuged at1200 rpm for 10 minute. The media was aspirated and the cell pelletresuspended in 15 ml Binding Media. The cells were centrifuged again andresuspended in the appropriate amount of Binding Media to be used forassays (50 ul of cells per well at 2× the final density). To each well,and equal volume (50 ul) of test compound was added and the plate wasincubated for 1.5 hours at 37 C, 5% CO2. Each well was rinsed 3× with150 ul per well of Binding Media. CyQuant NF reagent was prepared assuggested by manufacturer), and 100 ul of CyQuant NF reagent was addedper well. The plate was incubated at 37 C, 5% CO2, for 45 minutes. Theplate was protected from light by using black adhesive seals.Fluorescence intensity was measured using a Molecular Devices Gemini EMFluorescent Plate Reader (Ex 485/Em530, Bottom Read, ReadingSensitivity=20). IC50 curves are generated using Graph Pad Prism and thecurves analyzed using analyzed using a non-linear regression (fourparameters) algorithm. The log (concentration) versus RFU (Ex485/Em530)was plotted to determine IC50 values.

α4β7-MAdCAM Cell Adhesion Assay

RPMI 8866 cells (Sigma #95041316) were cultured in RPMI 1640 HEPESmedium (Invitrogen #22400-089) supplemented with 10% serum (Fetal BovineSerum, Invitrogen #16140-071), 1 mM sodium pyruvate (Invitrogen#11360-070), 2 mM L-glutamine (Invitrogen #25030-081) andPenicillin-Streptomycin (Invitrogen #15140-122) at 100 units ofpenicillin and 100 μg of streptomycin per ml. The cells were washed twotimes in DMEM medium (ATCC #30-2002) supplemented with 0.1% BSA, 10 mMHEPES pH 7 and 1 mM MnCl₂. The cells were re-suspended in supplementedDMEM medium at a density of 4×10⁶ cells/ml.

A Nunc MaxiSorp plate was coated with rh MAdCAM-1/Fc Chimera (R&D#6065-MC) at 200 ng per well in 50 ul per well in 1×PBS and incubated at4° C. overnight. The solution was then removed by shaking, blocked with250 ul per well PBS containing 1% BSA, and incubated at 37° C. for 1 hr.The solution was removed by shaking. Peptides were diluted by serialdilution in a final volume of 50 ul per well (2× concentration). To eachwell, 50 ul of cells (200,000 cells) were added and the plate wasincubated at 37° C., 5% CO₂ for 30-45 min to allow cell adhesion. Thewells were washed manually three times (100 ul per wash) withsupplemented DMEM. After the final wash, 100 ul/well of supplementedDMEM and 10 ul/well of MTT reagent (ATTC cat#30-1010K) were added. Theplate was incubated at 37° C., 5% CO2 for 2-3 hrs until a purpleprecipitate is visible. 100 ul of Detergent Reagent (ATTC cat#30-1010K)was added to each well. The plate was covered from the light, wrapped inParafilm to prevent evaporation, and left overnight at room temperaturein the dark. The plate was shaken for 5 min and the absorbance at 570 nmis measured. To calculate the dose response, the absorbance value ofcontrol wells not containing cells was subtracted from each test well.

α4β1-VCAM Cell Adhesion Assay

Jurkat E6.1 cells (Sigma #88042803) were cultured in RPMI 1640 HEPESmedium (Invitrogen #22400-089) supplemented with 10% serum (Fetal BovineSerum, Invitrogen #16140-071), 1 mM sodium pyruvate (Invitrogen#11360-070), 2 mM L-glutamine (Invitrogen #25030-081) andPenicillin-Streptomycin (Invitrogen #15140-122) at 100 units ofpenicillin and 100 μg of streptomycin per ml. The cells were washed twotimes in DMEM medium (ATCC #30-2002) supplemented with 0.1% BSA, 10 mMHEPES pH 7 and 1 mM MnCl₂. The cells were re-suspended in supplementedDMEM medium at a density of 4×10⁶ cells/ml.

A Nunc MaxiSorp plate was coated with rh VCAM-1/CD106 Fc chimera (R&D#862-VC) at 400 ng per well in 50 ul per well in 1×PBS and incubated at4° C. overnight. The solution was then removed by shaking, blocked with250 ul per well PBS containing 1% BSA, and incubated at 37° C. for 1 hr.The solution was removed by shaking. Peptides were diluted by serialdilution in a final volume of 50 ul per well (2× concentration). To eachwell, 50 ul of cells (200,000 cells) were added and the plate wasincubated at 37° C., 5% CO₂ for 30-45 min to allow cell adhesion. Thewells were washed manually three times (100 ul per wash) withsupplemented DMEM. After the final wash, 100 ul/well of supplementedDMEM and 10 ul/well of MTT reagent (ATTC cat#30-1010K) were added. Theplate was incubated at 37° C., 5% CO2 for 2-3 hrs until a purpleprecipitate is visible. 100 ul of Detergent Reagent (ATTC cat#30-1010K)is added to each well. The plate was covered from the light, wrapped inParafilm to prevent evaporation, and left overnight at room temperaturein the dark. The plate was shaken for 5 min and the absorbance at 570 nmis measured. To calculate the dose response, the absorbance value ofcontrol wells not containing cells was subtracted from each test well.

The potency, selectivity and stability data for certain illustrativepeptide monomers and dimers of the present invention are provided inTables 6 and 7. These peptides have the structures shown in Tables 4 and5, which may be identified by their SEQ ID NOs. Table 6 providespotency, selectivity and stability data for representative peptidemonomers. Table 7 provides potency, selectivity and stability data forrepresentative peptide dimers. For potency, IC50 values are shown as*<25 nM **=25-100 nM, ***=100-1000 nM. Where data not shown, data wasnot determined, but is expected that these peptides have an IC50<100 nMin α4β7 ELISA and/or cell assays.

TABLE 6 Characterization of Illustrative Thioether Monomer Peptides SEQID ELISA ELISA Cell-Adhesion PBMC SIF (Porcine) SGF (Porcine) NO A4B7(nM) A4B1 (nM) A4B7 (nM) IC50 (nM) (half-life, min) (Half-life, Min) 49** >1000 50 *** 51 ** 6 52 >1000 53 *** >180 54 >1000 second 55 * ****** 25 56 * *** *** 186 57 *** <20 58 >1000 59 *** <20 60 * *** >18061 * >180 62 * >180 63 * >180 64 * 179 65 * ** >180 66 ** >180 67 * <2068 * >180 69 * *** >180 70 * >180 71 ** >180 72 * >180 73 * >18074 * >180 75 * >180 76 * >180 77 * 88 78 * 78 79 * 80 * ** 81 * *** **82 * *** 83 * 84 * 85 * 86 * 87 * 88 * ** 89 * *** 90 * 91 * *** 92 * **93 * ** 94 * *** >180 95 * *** >180 96 * *** 26 97 * ***** >180, >180 >180 98 * *** ** *** >300 >180 99 * 100 * ** 101 * *** ***102 * ** 103 * 104 * ** 105 * 106 * ** 107 * ** 108 * ** 109 * 110 * **111 >1000 112 >1000 113 * ** 114 * ** ** >180 115 * 116 * ** 117 *** 118*** 119 *** 120 *** 121 *** 122 *** 123 ** 124 ** 125 * *** >180 126** >180 127 ** 128 ** 129 ** 130 *** 131 ** 132 * *** >180 133 **** >180 134 * ** >180 (428) 135 * *** 136 ** 137 ** 138 *** 139 **140 >1000 141 >1000 142 *

TABLE 7 Characterization of Illustrative Thioether Peptide Dimers SEQELISA Cell ID ELISA A4B1 Cell-Adhesion Adhesion PBMC SIF (Porcine) SGF(Porcine) NO A4B7 (nM) (nM) A4B7 (nM) A4B1 (nM) IC50 (nM) (half-life,min) (Half-life, Min) 143 * >1000 *** 144 * >1000 >1000 <20 145 *** **146 * ** * <20 147 >1000 148 >1000 149 >1000 <20 150 *** * >100,000 >180, >180, >180 >300 151 * ** * >180 152 *** * >100,000 >180 >60 153 *** * >180 (275) 154 * ** * >100,000 <20155 * *** * ** >180, >300 >180 156 ** ** >100,000 >180 157 *** ** <20158 * *** * >100,000 >180 159 * ** * >100,000 >180 >180 160 * * >180 >60161 * ** * >100,000 >180 162 *** >180 163 * >180 164 *** * >100,000 >180 >60 165 * ** * >100,000 >180 >60 166 * ** * 30 167 *** * >100,000 <20 168 * ** * >100,000 169 **** * >100,000 >180, >180 >180 170 * 171 * 172 >1000 173 ** 174 * 175 *176 * 177 * 178 * *** * >1000 >180 (375), >180 >180 (266), >180 179 ***180 *** 181 >1000 182 ** 183 ** 184 * *** * >100,000** >180, >180, >180 >180 185 ** 186 ** 187 ** 188 >1000 189 >1000 190 *191 * >100,000 192 * 193 * 194 *** * 195 * 196 * 197 * 198 * 199 * 200 *201 * 202 *** 203 *** 204 * *** * >100,000 >180 >180 205 *** 206 >1000207 * *** * >100,000 >180 >180 208 * ** * >100,000 >180 (312) >180 209 **** * >100,000 >180 210 *** 211 * 7 212 ** 213 * >180 (419) 214 ** 215 *216 * >180 217 ** 218 * 219 * >180, 407 >360 220 * >180 221 ** 222 * *223 * *

TABLE 8 Characterization of Illustrative Peptide Monomers ELISA Cell-SIF Redox SEQ ID Peptide A4B7 ELISA Adhesion (Porcine) stability NOsequence 1 2 3 4 5 6 7 8 9 10 (nM) A4B1 (nM) A4B7 (nM) (half-life, min)(DTT) 385 Ac C R S D T L C G E NH2 97 2020 590 <1 min ~3 min 386 Ac C RS D T L C NH2 96.8 2880 1221 <1 min ~3 min

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

The present invention may be embodied in other specific forms withoutdeparting from its structures, methods, or other essentialcharacteristics as broadly described herein and claimed hereinafter. Thedescribed embodiments are to be considered in all respects only asillustrative, and not restrictive. The scope of the invention is,therefore, indicated by the appended claims, rather than by theforegoing description. All changes that come within the meaning andrange of equivalency of the claims are to be embraced within theirscope.

1-19. (canceled)
 20. A peptide molecule comprising a structure ofFormula (VI):Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹   (Formula VI)or a pharmaceutically acceptable salt thereof, wherein Xaa¹ is a2-Me-benzoyl group capable of forming a thioether bond with Xaa⁷; Xaa²is selected from the group consisting of N(alpha)-Me-Arg, Arg, HArg,Dap, Dab, Arg-Me-sym, Arg-Me-asym, 4-Guan, Cit, Cav, and suitableisostere replacements; Xaa³ is selected from the group consisting ofSer, Gly, and suitable isostere replacements; Xaa⁴ is selected from thegroup consisting of Asp, N-Me-Asp, Asp(OMe), D-Asp, and a suitableisostere replacements; Xaa⁵ is selected from the group consisting ofThr, Gln, Ser, Asp, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Asn, Glu,Val, Tyr, Trp, Leu, Met, and N-Methyl amino acids including N-Me-Thr,and suitable isostere replacements; Xaa⁶ is selected from the groupconsisting of Gln, Asn, Asp, Pro, Gly, Ala, Phe, Leu, Glu, Ile, Val,HLeu, n-Butyl Ala, n-Pentyl Ala, n-Hexyl Ala, Nle, cyclobutyl-Ala,N-Me-Leu, and suitable isostere replacements; Xaa⁷ is selected from thegroup consisting of Cys, N-Me-Cys, D-Cys, HCys, Pen, and D-Pen; Xaa⁸ isselected from the group consisting of absent, Gly, Gln, Asn, Asp, Ala,Ile, Leu, Val, Met, Thr, Lys, Trp, Tyr, His, Glu, Ser, Arg, Pro, Phe,Sar, 1-Nal, 2-Nal, HPhe, Phe(4-F), O-Me-Tyr, dihydro-Trp, Dap, Dab,Dab(Ac), Orn, D-Orn, N-Me-Orn, N-Me-Dap, D-Dap, D-Dab, Bip,Ala(3,3diphenyl), Biphenyl-Ala, aromatic ring substituted Phe, aromaticring substituted Trp, aromatic ring substituted His, hetero aromaticamino acids, N-Me-Lys, N-Me-Lys(Ac), Bpa, Phe(3-Me), Phe(2-Me),Phe(2-CF3), β-Me-Phe, 4-Me-Phe, and corresponding D-amino acids andsuitable isostere replacements; Xaa⁹ is selected from the groupconsisting of absent, Glu, Amide, Lys, COOH, CONH₂, Gln, Pro, Gly, His,Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Gla, Ser, Asn, D-Glu,β-HGlu, 2-Nal, 1-Nal, D-Asp, Bip, β-HPhe, β-Glu, D-Tyr, D-Lys, Dap, Dab,Orn, D-Orn, N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me Lys, D-Dap, D-Dab, Glu,N-Me-Asp, alpha-H-Glu, suitable isosteres, and corresponding D-aminoacids; Xaa¹⁰ is selected from the group consisting of absent, Gln, Pro,Gly, His, Ala, Ile, Phe, Lys, Arg, Leu, Val, Tyr, Trp, Met, Glu, Ser,Asn, Gla, Dap, Dab, Orn, D-Orn, D-Lys, N-Me-Orn, N-Me-Dap, N-Me-Dab,N-Me-Lys, D-Dap, D-Dab, COOH, CONH₂, suitable isosteres, andcorresponding D-amino acids; and Xaa¹¹ is selected from the groupconsisting of absent, Gln, Pro, Gly, His, Ala, Ile, Phe, Lys, Arg, Leu,Val, Tyr, Trp, Met, Glu, Ser, Asn, Gla, Dap, Dab, Orn, D-Orn, D-Lys,N-Me-Orn, N-Me-Dap, N-Me-Dab, N-Me-Lys, D-Dap, D-Dab, COOH, CONH₂,suitable isosteres, and corresponding D-amino acids, wherein the peptidefurther comprises a thioether bond between Xaa¹ and Xaa⁷, wherein thepeptide further comprises a thioether bond between Xaa¹ and Xaa⁷. 21.The peptide molecule or pharmaceutically acceptable salt thereof ofclaim 20, wherein Xaa⁴ is a 2-methyl benzoyl moiety that forms athioether bond with Xaa¹⁰, Xaa⁵ is N-Me-Arg, Xaa⁶ is Ser, Xaa⁷ is Asp,Xaa⁸ is Thr, Xaa⁹ is Leu, and Xaa¹⁰ is Pen, Cys, D-Cys or HomoCys. 22.The peptide molecule or pharmaceutically acceptable salt thereof ofclaim 21, wherein Xaa¹⁰ is Pen or Cys.
 23. The peptide molecule orpharmaceutically acceptable salt thereof of claim 20, further comprisinga terminal modifying group selected from the group consisting of DIG,PEG4, PEG13, PEG25, PEG1K, PEG2K, PEG4K, PEG5K, Polyethylene glycolhaving molecular weight from 400 Da to 40,000 Da, IDA, Ac-IDA, ADA,Glutaric acid, Isophthalic acid, 1,3-phenylenediacetic acid,1,4-phenylenediacetic acid, 1,2-phenylenediacetic acid, AADA, suitablealiphatic acids, suitable aromatic acids, and heteroaromatic acids. 24.The peptide molecule of claim 20, wherein the C-terminus of the peptidemolecule further comprises a modifying group.
 25. The peptide moleculeor pharmaceutically acceptable salt thereof of claim 20, wherein thepeptide molecule is a monomer.
 26. The peptide molecule orpharmaceutically acceptable salt thereof of claim 20, wherein thepeptide molecule is a dimer.
 27. The peptide molecule orpharmaceutically acceptable salt thereof of claim 26, comprising twopeptide molecules dimerized by a linker.
 28. The peptide molecule orpharmaceutically acceptable salt thereof of claim 27, where in thelinker is selected from the group consisting of: DIG, PEG4, PEG4-biotin,PEG13, PEG25, PEG1K, PEG2K, PEG3.4K, PEG4K, PEG5K, IDA, ADA, Boc-IDA,Glutaric acid, Isophthalic acid, 1,3-phenylenediacetic acid,1,4-phenylenediacetic acid, 1,2-phenylenediacetic acid, Triazine,Boc-Triazine, IDA-biotin, PEG4-Biotin, AADA, suitable aliphatics,aromatics, heteroaromatics, and polyethylene glycol based linkers havinga molecular weight from approximately 400 Da to approximately 40,000 Da.29. The peptide molecule or pharmaceutically acceptable salt thereof ofclaim 27, wherein the two peptide molecules are dimerized via theirC-termini.
 30. A pharmaceutical composition comprising the peptidemolecule or pharmaceutically acceptable salt thereof of claim 20 and apharmaceutically acceptable carrier, diluent or excipient.
 31. Thepharmaceutical composition of claim 30, wherein the pharmaceuticalcomposition is formulated for oral delivery.
 32. The pharmaceuticalcomposition of claim 30, further comprising an enteric coating.
 33. Thepharmaceutical composition of claim 32, wherein the enteric coatingreleases the pharmaceutical composition within a subject's lowergastrointestinal system.
 34. A method for treating or preventing adisease or condition that is associated with a biological function ofintegrin α4β7, the method comprising providing to a subject in needthereof an effective amount of the peptide molecule or pharmaceuticallyacceptable salt thereof or the pharmaceutical composition of claim 20.35. The method of claim 34, wherein the disease or condition is selectedfrom the group consisting of Inflammatory Bowel Disease (IBD), adultIBD, pediatric IBD, adolescent IBD, ulcerative colitis, Crohn's disease,Celiac disease (nontropical Sprue), enteropathy associated withseronegative arthropathies, microscopic colitis, collagenous colitis,eosinophilic gastroenteritis, radiotherapy, chemotherapy, pouchitisresulting after proctocolectomy and ileoanal anastomosis,gastrointestinal cancer, pancreatitis, insulin-dependent diabetesmellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronicbronchitis, chronic sinusitis, asthma, primary sclerosing cholangitis,human immunodeficiency virus (HIV) infection in the GI tract,eosinophilic asthma, eosinophilic esophagitis, gastritis, colitis,microscopic colitis and graft versus host disease (GVHD).
 36. The methodof claim 34, wherein the disease or condition is an inflammatory boweldisease, ulcerative colitis, or Crohn's disease. 37-38. (canceled) 39.The method of claim 34 wherein the peptide molecule or pharmaceuticallyacceptable salt thereof inhibits binding of α4β7 to MAdCAM.
 40. Themethod of claim 34, wherein the peptide molecule or pharmaceuticallyacceptable salt thereof or the pharmaceutical composition is provided tothe subject in need thereof at an interval sufficient to ameliorate thecondition.
 41. (canceled)
 42. The method of claim 34, wherein thepeptide molecule or pharmaceutically acceptable salt thereof orpharmaceutical composition is provided as an initial does followed byone or more subsequent doses, and the minimum interval between any twodoses is a period of less than 1 day, and wherein each of the dosescomprises an effective amount of the peptide molecule orpharmaceutically acceptable salt thereof.
 43. The method of claim 34,wherein the effective amount of the peptide molecule or pharmaceuticallyacceptable salt thereof or the pharmaceutical composition is sufficientto achieve at least one of the following: a) about 50% or greatersaturation of MAdCAM binding sites on α4β7 integrin molecules; b) about50% or greater inhibition of α4β7 integrin expression on the cellsurface; and c) about 50% or greater saturation of MAdCAM binding siteson α4β7 molecules and about 50% or greater inhibition of α4β7 integrinexpression on the cell surface, wherein i) the saturation is maintainedfor a period consistent with a dosing frequency of no more than twicedaily; ii) the inhibition is maintained for a period consistent with adosing frequency of no more than twice daily; or iii) the saturation andthe inhibition are each maintained for a period consistent with a dosingfrequency of no more than twice daily.
 44. The method of claim 34,wherein the peptide molecule or pharmaceutically acceptable salt thereofis administered orally. 45-46. (canceled)